Lecture 1 Date: Sept. 10, 004 By Bhaweshwar Das Introduction: COURSE DETAILS

Section I Natural Resources of Nepal: Eco-climatic division of Nepal; The biodiversity and its Economic strength Species in trade: Plant genera in trade; Species in Export, import; & Re-trade As for example: Swertia, Nardostachys, Parmelia, Asparagus, Picrorhiza, Taxus, Daphne, Sapindus, Euphorbia, Zanthoxylum. Etc Challenges in commercialization of plant resources: Quality management [raw material to finished products; Opportunity with plant resources] Section II an Introduction of Natural Product Chemistry: Alkaloids, Terpenoids, Carotenoids, Anthocyanins, Flavonoids, Xanthones, and Steroids Section III Chemistry of twenty major species of trade [Species of Export 10, import 5; & Re-trade Alkaloids Anthraquinones, Catechins, Tannins, Glycosides, Terpenes, Xanthones, Section IV Isolation, purification and identification of active ingredients: Pulverization, Distillation, Solvent extraction, Solvent-solvent extraction, Crystallization and re-crystallization, T.L.C., Column chromatography; G.C., HPLC; Analysis of active ingredient of above selected Species: Application of modern analytical methods. M.P.; B.P.; refractive index, Optical rotation, Flash point, Co-TLC; G.C.; U.V.; I.R.; N.M.R.; ----------- [Species in trade: Plant genera in trade: Species in Export, import; & Re-trade As for example: Swertia, Nardostachys, Parmelia, Asparagus, Picrorhiza, Taxus, Daphne, Sapindus, Euphorbia, Zanthoxylum. etc] -------------- What are the gaps? Where shall we concentrate? Where to read?

Lecture 2 Date: September 12,2004

Session Topic: Eco-climatic division of Nepal: Ecology and climate of a place is governed by fact where the place is located.

1) Location: � Longitude � Latitude 2) Altitude from sea level 3) Mountains around 4) Ocean and sea around the place 5) Hot and cold water current of air and water

We are located in Asia between 80°03” E to 88°10” E and 26°30”N to 30°26”N. This geographical location encompasses a portion of Himalayan range in the north and gangetic plane in south. The specific location spreads its geographical territory on either side of Himalayan range. Districts west to Rasuwa like Manang, Mustang, Dolpa, Mugu, Humla lie in the north of Himalayas.

Because of its specific location on earth the country has wide altitudinal range. These mountains contribute a lot to climate of Nepal. The mountains are too cool some part even below -30°c; while the planes are much hotter some places above +30° c.

Max. Max. Min. Min. Rainfall Rainfall

Stations temp. (ºC) temp. (ºC)

temp. (ºC) temp. (ºC) (mm)# (mm)#

Dadeldhura 26.2 15.5 15.5 3.4 12.8 0

* Dipayal 38 25.5 22.8 4 0 0

Dhangadi 33 22 23 5.8 0 0

* Birendranagar 33.2 21.5 21.8 4.5 Traces 0

Nepalgunj 33.7 21.2 24.5 5.5 0 0

Jumla 26 13.1 12.5 -5.5 0 0

Dang 32.2 22.4 21.5 4 0 0

* Pokhara 31 22.6 22 7 22.5 0

Bhairahawa 34.9 22.6 26 6 0 0

Simra 32.5 26 22.5 6.5 7.2 0

* Kathmandu 30.2 20.5 20 3 0 0

Okhaldhunga 24.6 17.6 17.6 7.5 0 0

Taplejung 24.4 15.8 17.9 4.6 1.5 0

* Dhankuta 26.5 21.8 20.8 8.1 Traces 0

Biratnagar 34.2 27.5 25 7.8 12.8 0

Nagarkot 21.2 25.8 14 8.5 0.3 0

Jomsom 23 13.3 13 0 0 0

Bharatpur 35 0 19 0 10.5 NA

Climatic Information is based on HMG Department of Hydrology and Metrology.

The rain in Nepal is mainly due to monsoon, which originates in Bay of Bengal and passes all the way as lead by the Himalayan range. It is cooled due low temperature of mountains and precipitates as rain. These rains are heavy in the eastern Nepal and as monsoon moves west, it becomes water deficient and there is low rain in the western Nepal. These clouds are also blocked by the mountain range the area falls in rain shadow zone. This is why districts west to Rasuwa like Manang, Mustang, Dolpa, Mugu, Humla which lie in the north of Himalayas have low rain. These physiographic processes have created different climatic zones in Nepal. Altitude: Our geographical location

Belts Altitude

Sub-tropical climate Up to 1200m Warm temperature climate 1200-2000m Cool temperate climate 2100-3300m Alpine ‘Lekali’ climate 3300-5000m Himalayan desert climate Above 5000m

Lecture 3

September 17, 2004

CLIMATE TYPES AND ITS SPECIFIC FEATURE:

1. Sub-tropical climate:

The climate is seen in southern planes of the country earth at present, which has an altitude between 60m to 1200m. They include Terai, Bhawar, Chure, and inner terai; it can also be observed in Besi, Tar, and khonch. Some part of the country with low altitudes is too hot, while others are humid semitropical climate. This climate is found up to 1200m. Climatologically speaking this is sub-tropical but for Nepal, this is tropical. Summer temperature in this zone ranges from 25°c to 40°c. The climate has hot waves in summer. Western terai districts like Kanchanpur, Kailali, Bardiya, Banke, valleys of Dang and Surkhet, Kapilbastu, Rupandehi are among hot compared to eastern districts like Saptari and Morang and Jhapa. Rain is due to monsoon, which originates in the Bay of Bengal and enters from east form where it extends to west where upon it gradually desiccates and western terai has low rain in summer. When the monsoon returns from Hinudkush in the winter, the winter rain prevails. The winter rain is more in west than in the east. The terai has over 200mm rain. Accacia, Adina, Alastonia, Annona, Bombax, Bamboo, cassia, ceaselpinea, Cinnamomum, Eagle, Ficus, Hollorhena, Mallotus, melia, Nerium, Oroxylon, Phyllanthus, Sapindus, Shorea, Dalbergia, Strychnos, Tamarind, Terminalia are the major the major tree genera of this climate, while Mucuna, Dioscorea, Gloriosa, Gymnema are the major climbers in this climate. Adhatoda, Basil, Cassia, Lantana, Pogostemon, Pandanus constitute the major shrubs and Acorus, Amorphophyllus, Andrographis, Asparagus, Bacopa, Boerhavia, Eclipta, Eulophia, Phyllanthus Swertia, Vinca, Vetiver Withania are some of the typical herbs found in this climate.

2. Warm temperature climate: This climate prevails in the Mahabharat range from 1200m to 2100 m .The summer temperature is ranges between 24°c to 30°c while the winter temperature falls up to 0°c depending on altitude. River basins are hot and humid. The valleys like Kathmandu fall in a climate where summer days reaches up to 33°c and cool winter night are as low as minus 3°c. In the southern face of the Mahabharat range, there is heavy rain up to 125 cm, while on the northern side of the range the rain is relatively low only up to 100 cm. Accacia, Adina, Bombax, Bamboo, cassia, Cinnamomum, Eagle, Ficus, Mallotus, Phyllanthus, Sapindus, Terminalia are the major the major tree genera of this climate, while Mucuna, Dioscorea, are the major climbers in this climate. Basil, Cassia, Lantana, Pogostemon, constitute the major shrubs and Acorus, Asparagus, Swertia, Vinca, are some of the typical herbs found in this climate. This is the climate range where most of the mountainous caste lives.

3. Cool temperate climate: The Himalayan climate above 2100m to 3300m is quite contrasting to tropical climate of the terai. Various districts that lie between south of Himalayas and Mahabharat range are in this climate zone. They include Taplejung, Panchthar, Sankhuwasbha, Ramechap, Dolkha, in the East and Rasuwa, Dhadhing, Gorkha, Lamjung, Parbat in the west represent such districts. With the exception of river basin with low altitude and small valley in the range the whole of the above districts are in cool temperate climate zone.

The summer temperature in these districts ranges between 15°c to 20°c. They have near zero°c temperature in the winter. They have snowfall in the winter. Rain is mainly due to monsoon but only up to 100 cm. The rain is high in the south and in the east and comparatively lower in the north and west. Districts in this climate zone have cool and windy days and nights in the winter. The tree species present are Pinus, Rhododendron, Quercus, Daphnephyllum, Abies, and Tsuga Among the Medicinal plant, several important traded species of Nepal such as Rhubarb, Aconitum, Asparagus, Gaultheria, and Pseudo-panax are collected from this climate zone.

4. Alpine ‘Lekali’ climate: The Himalayan range above 3300m to 5000m is in this climate zone. This climate is similar to climate of Alps in Europe and there fore called alpine climate. The nine-month of the year is snow laden. The snow melts in summer months. The summer temperature ranges between 10°c to 15°c. The Rain is below 40 cm. Tree species of the zone are Cedrus, Junipers, Cuperessus, Larix, Hipophae, and Taxus. Some of the high value medicinal plants like Cordyceps, Aconitum, Picrorhiza; Nardostachys, Ephedra, Onsoma, Rheum, Carum, are collected from this climate zone.

5. Himalayan desert climate: The altitude above 5000m i.e. above snowline is laden with snow all the year round and are not suitable for any vegetation not even lichen can grow here. Because the typically ice laden characters round the year like polar region it is also called third pole.

The evolution of earth: The earth was formed from a solar cloud of Hydrogen under gravitation. Earth’s first rock was formed over 3500 million year ago. Different continent of the earth was a single landmass by that time. This single landmass; according to continental drift theory of Alfred Wagener (1915) split into Laurasia and gondwana land. The Gondwana land further split into India, Antarctica and Australia.

EVOLUTION OF EARTH

The sediments of shallow sea “Tethys Sea” between Africa and Tibetan plateau raised due to drift of India and Africa northward. The Himalayan range emerged due to this drift. A radical idea of plate tectonics assumes that the earth is made of series of rigid crusts called plates. These plates are moved by convection current within the surface of earth. These plates diverge and converge along margins, which is marked by earthquakes and volcanoes. Molten lava within pushes plates up, by which mountains and ranges are formed. The soil: This formation of mountains and ridges took of years. The rain and other denudation processes went on. The soft-top soils were carried and deposited at riverbeds. The terai of to day is made up of such alluvial soils brought and deposited by river Ganges and its sisters. Moreover, whole of terai except Dang, and Chitwan is made up of such alluvial soils.

Geological map of Nepal

Sub Himalayan basin /dun of northern Dang, Northern rauthat, Northern Sarlahi is made of Alluvial soils brought by respective rivers. Lesser Himalayan Fluvial soils of Kathmandu valley and northwestern Tanahun are conglomerates and lacustrine clay. Lower part of sivaliks in Kanchanpur, Kailali, and peripheral Dang is made up of marine shells and limestone and fluvial soils. South of Salyan, Rolpa, Pyuthan, North of Palpa, Nawalaparashi are mainly shallow marine sediments phyllites, sandstone, and quartzite. Soils of Myagdi, Kaski, and Lamjung are flyschoid sequences of bedded schist, phyllites and Meta sandstone. Several mountain districts are rich in high-grade metamorphic rocks; these districts include Dolkha, north Ramechap, Solu and other eastern districts. Thus, what we see that the soils of Nepal are made up of various types of rocks, which in course of time denudated contributed to the formation of different soil structures of the country. The difference lies not only in names of its component rocks but also in its chemical compositions and presence or absence of micro-components like individual elements. They also contribute to water holding capacity of the soils. The soil composition has immense impact on growth or flourishing of individual plant species.

If a soil structure is suitable for a species of plant, it may or may not provide enough nutrients to other plant species. Lecture 4 September 24, 2004 BIODIVERSITY Biodiversity = Bio [= plants animals] + diversity [= variances, multiplicity, difference, dissimilarity]

� Ecosystem level � Species level � Genetic level [Chemo-type level]

Ecosystem level: In our earlier lectures, we talked on how the physiography of Nepal has significant impact on its climates; and how the origin of the mountain ranges and river-system created different soil condition within small area of the country.

These differences in climate, water availability, soil composition and the soil moisture, create different ecosystem in the different part of the country. Stainton (1972) classified the country in to seven different ecosystem as follows:

1. Terai and Bhawar. 2. Siwalik, and Dun valley. 3. Mahabharat. 4. Mid lands 5. Himalyas. 6. Inner Himalayas 7. Arid zones

1. Terai and Bhawar. Southern part of the country adjoining Indian Territory in the south and siwalik range in the north is called Terai. Its altitude varies from 60 m from sea level to 300 m. Forest zone called “charkose jhari” in the area was once very rich and diverse in tropical plant diversity.

2. Siwalik, and Dun valley. This lies between north of terai and south of Mahabharat

range.The altitude of this ecosystem is between 300 m to 1500 m. The range is wide in the western Nepal and less wider in the east. Chitwan,Dang and trijuga are inner terai valley of Nepal. They are sloppy and fertile.

3. Mahabharat. Mahabharat is the range spread east to west in north of Siwalik and south of

mid land. The altitudes ranges from 1500 m to 2700 m. River like Bagmati, Babai flow through it.

4. Mid lands: this ecosystem prevails in the north of Siwalik and Mahabharat and south of

Himalyas. Altitude ranges from 600m to 3500 m. Kthmandu, Pokhara, Banepa are located in this zone. This is densely populated next to terai. The climate here is more appropriate and land is fertile.

5. Himalyas. High mountains like Saipal, Lhotse, Machpuchre, Annapurna, Choyu, Manaslu, Makalu, , Sagarmatha, Kanchanjungha range are located here. The altitudes above 5500 m are snow laden and are devoid of flora.

6. Inner Himalayas: The trans Himalayan districts like Humla, Jumla, Manang, Mustng, can also be considered inner Himalayas. Plants species like Taxus, Abies, Tsuga, and Rhododendron are found here.

7. Arid zones: Some part of Dolpa, Manang, and Mustang are part of Tibetan plateau and the climate here is arid.

Species level:

Living beings capable of breeding mutually are called species. Species diversity means number and frequency of different species in an area. The earth is expected to in habit from 5,000000 to 3 0,000000 species of living beings (Wilson, 1988).

Richness in diversity means total number of different species in particular area while The term relative abundance indicates availability of one species compared to the other. The dominant species means the species which out numbers other species. Example cited from Class / village. Community forest where tree species having timber values are kept and other bushy and thorny species are taken out cannot be considered rich in diversity. Thus, Richness in diversity implies proportionate growth of all potential species in the area.

Plant Species in the World

Species in Nepal

Compared to world [%]

Lichen 20000 465 2.3 Fungi 69000 1822 2.6 Algae 26900 687 2.5 Mosses /liver. 16600 853 5.1 Pteridophytes 11300 380 3.4 Gymnosperm 529 27 5.1 Angiosperm 220000 58600 2.6

These different species are sources of different chemical example

Genetic level:

Gene is the smallest entity present in DNA that carries parental character to the progeny. Genetic diversity indicates differences in arrangement of genes in the chromosomes. This leads to differentiation of brothers and sisters from among their sisters. Example: there are different types of rice in Nepal such as Kuriya, Basmati, Anandi, Thapchininya, Marsi, Chomrong etc. This not only creates morphological variances among the progenies but also its content and composition. To a chemist the genetic variation is significant because it differentiates the chemical composition from among the plant of same species. Example:

ARTEMISIA SPECIES IN NEPAL

1. Artemisia albida Willd.ex Ledeb.

2. Artemisia biennis Willd.

Lecture 5 September 26, 2004 We have seen that in a small area of Nepal we have quite good number of plant species as high as 62834 plants (so far known), stay alive. These species are in continuous threat of extinction due to changes in ecology, Climate and human intervention. In the late twentieth century, it was realized that if no attention is paid on these changes, existence of future generation will be impossible. . Convention of Biodiversity

At the 1992 Earth Summit in Rio de Janeiro, world leaders agreed on a comprehensive strategy for "sustainable development" -- meeting our needs while ensuring that we leave a healthy and viable world for future generations. One of the key agreements adopted at Rio

3. Artemisia caurifolia

4. Artemisia capillaris Thunb.

5. Artemisia dubia Wall. 6. = vulgaris

7. Artemisia gmelinii var. vestita (Wall.ex DC.) = Artemisia vestita (Wall.ex DC.)

Artemisia gmelinii var. minor (Ledeb.) = Artemisia sacrorum Ledeb.

8. Artemisia indica =vulgaris Artemisia indica var. canadensis Besser Artemisia indica var. orientalis (Pamp.) H.Hara Artemisia indica Willd.

9. Artemisia japonica Thunb.

10. Artemisia juncea Kar. & Kir.

11. Artemisia maritima Linn.

12. Artemisia moorcroftiana Wall.

13. Artemisia myrianth

14. Artemisia parviflora Buch.-Ham.ex Roxb.

15. Artemisia roxburghiana Bess.

16. Artemisia siversiana Ehrh.ex Willd. 17. Artemisia vulgaris Burm.f. = indica

18.

was the Convention on Biological Diversity. This pact among the vast majority of the world's governments sets out commitments for maintaining the world's ecological underpinnings as we go about the business of economic development. The Convention establishes three main goals:

1. Conservation of biological diversity, 2. Sustainable use of its components, 3. Fair and equitable sharing of the benefits from the use of genetic resources;

Representatives of 155 countries agreed this document to achieve the common goal of sustainable development. There are 21 articles the document. These articles are further sub articles. These article explain the process by which the world can achieve the above goals.

International Union of Conservation Nature [IUCN] Nepal enlisted all the plant and animal species, which are under threat or at the verge of extinction in its “red list”. It has also published some books such as “Rare and endangered plants of Nepal” and "National Register of Medicinal plants " second edition. These books enlist plants, which are under threat, and especially the "National Register of Medicinal plants " second edition tried to enlist are which has been traditionally used in Nepal. The book claims to serve the purpose of Article 15,16 and 19 of “Convention Of Biological Diversity [CBD]”. Hence, we have tried to aware our colleagues on CBD Article 15,16 and 19.

Article 15 Access to genetic resources 1. Recognizing the sovereign rights of States over their natural resources, the authority to determine access to genetic resources rests with the national governments and is subject to national legislation. 2. Each Contracting Party shall endeavor to create conditions to facilitate access to genetic resources for environmentally sound uses by other Contracting Parties and not to impose restrictions that run counter to the objectives of this Convention. 3. For the purpose of this Convention, the genetic resources being provided by a Contracting Party, as referred to in this Article and Articles 16 and 19 are only those that are provided by Contracting Parties that are countries of origin of such resources or by the Parties that have acquired the genetic resources in accordance with this Convention. 4. Access, where granted, shall be on mutually agreed terms and subject to the provisions of this Article.

5. Access to genetic resources shall be subject to prior informed consent of the Contracting Party providing such resources, unless otherwise determined by that Party. 6. Each Contracting Party shall endeavor to develop and carry out scientific research based on genetic resources provided by other Contracting Parties with the full participation of, and where possible in, such Contracting Parties. 7. Each Contracting Party shall take legislative, administrative or policy measures, as appropriate, and in accordance with Articles 16 and 19 and, where necessary, through the financial mechanism established by Articles 20 and 21 with the aim of sharing in a fair and equitable way the results of research and development and the benefits arising from the commercial and other utilization of genetic resources with the Contracting Party providing such resources; Such sharing shall be upon mutually agreed terms. Article 16: Access to and transfer of technology 1. Each Contracting Party, recognizing that technology includes biotechnology, and that both access to and transfer of technology among Contracting Parties are essential elements for the attainment of the objectives of this Convention, undertakes subject to the provisions of this Article to provide and/or facilitate access for and transfer to other Contracting Parties of technologies that are relevant to the conservation and sustainable use of biological diversity or make use of genetic resources and do not cause significant damage to the environment. Lecture 6 2. Access to and transfer of technology referred to in paragraph 1 above to developing countries shall be provided and/or facilitated under fair and most favorable terms, including on concessional and preferential terms where mutually agreed, and, where necessary, in accordance with the financial mechanism established by Articles 20 and 21. In the case of technology subject to patents and other intellectual property rights, such access and transfer shall be provided on terms, which recognize and are consistent with the adequate and effective protection of intellectual property rights. The application of this paragraph shall be consistent with paragraphs 3, 4 and 5 below. 3.Each Contracting Party shall take legislative, administrative or policy measures, as appropriate, with the aim that Contracting Parties, in particular those that are developing countries, which provide genetic resources are provided access to and transfer of technology which makes use of those resources, on mutually agreed terms, including technology protected by patents and other intellectual property rights, where necessary, through the provisions of Articles 20 and 21 and in accordance with international law and consistent with paragraphs 4 and 5 below. 4. Each Contracting Party shall take legislative, administrative or policy measures, as appropriate, with the aim that the private sector facilitates access to, joint development and transfer of technology referred to in paragraph 1 above for the benefit of both governmental

institutions and the private sector of developing countries and in this regard shall abide by the obligations included in paragraphs 1, 2 and 3 above. 5. The Contracting Parties, recognizing that patents and other intellectual property rights may have an influence on the implementation of this Convention, shall cooperate in this regard subject to national legislation and international law in order to ensure that such rights are supportive of and do not run counter to its objectives. Article 19: Handling of biotechnology and distribution of its benefits 1. Each Contracting Party shall take legislative, administrative or policy measures, as appropriate, to provide for the effective participation in biotechnological research activities by those Contracting Parties, especially developing countries, which provide the genetic resources for such research, and where feasible in such Contracting Parties. 2. Each Contracting Party shall take all practicable measures to promote and advance priority access on a fair and equitable basis by Contracting Parties, especially developing countries, to the results and benefits arising from biotechnology based upon genetic resources provided by those Contracting Parties. Such access shall be on mutually agreed terms. 3. The Parties shall consider the need for and modalities of a protocol setting out appropriate procedures, including, in particular, advance informed agreement, in the field of the safe transfer, handling and use of any living modified organism resulting from biotechnology that may have adverse effect on the conservation and sustainable use of biological diversity. 4. Each Contracting Party shall, directly or by requiring any natural or legal person under its jurisdiction providing the organisms referred to in paragraph 3 above, provide any available information about the use and safety regulations required by that Contracting Party in handling such organisms, as well as any available information on the potential adverse impact of the specific organisms concerned to the Contracting Party into which those organisms are to be introduced. NTFP policy 2061, has recently been released by HMG Department of Plants Resources [Kantipur Bhadra 2061] CITES AND NTFPs OF NEPAL The Convention on International Trade in Endangered Species of Wild Fauna and Flora, or CITES, came into effect in 1975. The United Nations Environment Program administers the Convention and 150 countries are a Party to the Convention. The Convention was established with the aim of controlling and monitoring the international trade in plants and animals considered being threatened, or likely to become threatened, and affected by such trade. Generally, the Convention does not prohibit the international trade in

listed species, but seeks to prevent the unsustainable exploitation of wild populations of plants and animals. The Convention consists of a text setting out the main "rules" and three Appendices, or lists, of plants and animals. Trade in species included in these Appendices is controlled and monitored by means of a permit system. In CITES terms, "trade" means movement across international borders. Appendix: 1 All parts and derivatives subject to control, including live plants, hybrids, seeds, and herbarium and spirit material. 1.Saussurea costus 2.Vanda coerulea Appendix 2 All parts and derivatives subject to control, including live plants, hybrids, herbarium and spirit material, but not seeds (except seeds of Mexican Cactaceae originating from Mexico), spores and pollen (including pollinia), in vitro cultures unless otherwise annotated. 1. Aloe spp. 2. Cyathea spp. 3. Cycadaceae spp. 4. Dioscorea deltiodea 5. Euphorbia spp. 6. Hedychium philippinense 7. Nardostachys grandiflora 8. Orchidaceae spp. 9. Panax ginseng [Panax pseudoginseng]* 10. Picrorhiza kurroa* 11. Podophyllum hexandrum* 12. Pterocarpus santalinus 13. Rauvolfia serpentina 14. Taxus wallichiana Appendix 3 Controls = As appendix 2 1. Gnetum montanum 2. Magnolia liliifera var. obovata 3. Meconopsis regia 4. Podocarpus neriifolius 5. Tetracentron sinense

Lecture 7 October 3, 2004 Economic Strength of Bio-diversity Again we will limit our self to plant diversity because the animal are not the primary sources

of secondary metabolite and also because by definition secondary metabolites are products of plant origin.

� Past trade � Present trade � Future trade

Past trade: In the past, several species of plants were lost due to human intervention and popular use of plants. To enlist few of them here: Santalum album Pterocarpus santalianus Aqularia agalocha Strychnos nuxvomica Many others have become sparse

Melia indica, Ficus hamiltony, and perhaps several species of Ficus like religiosa, benghalensis, and Eagle marmelos, Madhuca butyracea, Alstonia scholaris, which are, now listed, as religious plants can be included in this list. Several of these species are sources of novel secondary metabolites. Present day trade: Lecture 8 The list of plants that are in the revenue list of H.M.G. department of Forest and published in Forest act 049 and Forest regulation 051 can be considered as sources of secondary metabolites that are traded from Nepal. Table below shows list plants that are in the revenue list: VOLUME OF MAP COLLECTED [DATA PUBLISHED BY MOFSC] NTFPs collected 056/057 East Central Western

Mid-west

Far-west Total

Allo 0 0 Amarisho 0 Amla 100 4394 0 300 4794 Argeli 0 11827 0 0 0 11827 Atish jara 0 0 848 1683 0 2531 Babiyo 0 0 Bair 0 500 0 0 0 500 Ban karaila bia 0 4610 87 0 0 4697 Bel 4000 0 0 0 0 4000 Bet ko phal 0 0 0 19365 0 19365 Bhadraksha 0 14254 0 0 0 14254 Bhoja patra 0 0 0 0 2529 2529 Bhorla ko bokra 0 7200 0 15000 12232 34432 Bhutkesh 0 681 0 0 2200 2881 Bhyakur 0 0 465 0 3104 3569 Bilauni 0 0 1450 150 0 1600 Bish jara 3670 3631 1625 1652 0 10578 Bish phenj 0 442 0 1016.5 0 1458.5 Bojho 3100 2270 1979 2885 1866 12100 Budhani 0 200 800 0 0 1000 Chabo 14752 0 0 0 0 14752 Champabati 0 444 0 0 0 444 Chiaraito 64387 62834.8 14216 24454 3470 169361.8 Choya bokra 0 0 0 0 200 200 Chyau 0 0 0 3270.5 33 3303.5 Dalchini 0 0 2586 13585 0 16171 Daru haldi 0 0 0 172 3500 3672

Dhupi pat 0 10684 1875 8557 200 21316 Gamdol 0 1910 353 220 0 2483 Ghortapre 0 2150 0 0 0 2150 Gobresalla ko pat 0 5000 0 0 0 5000 Gurjo 0 2227.5 0 0 2227.5 Harro 0 7979 0 0 0 7979 Inreni biu 1000 0 0 0 0 1000 Jatamanshi 0 3752 6224 72914 700 83590 Jhyau 700 40392 16090 83624.5 91871 232677.5 Jivanti 0 9625 0 0 0 9625 Kachur 0 0 0 0 32 32 kainyo phul 0 16030 0 0 0 16030 Kakar sighi 0 0 2958 0 0 2958 Kakoli jara 0 3348 0 800 0 4148 Kaladana 0 0 0 7381 Kantakari phul 3950 0 0 0 0 3950 Kara chulthi/amalbet 0 0 10358 3769 0 14127 Khair 0 Khoto 0 Kukur tarul 6771 0 0 0 6771 Kumkum pat 0 29600 0 0 0 29600 Kurilo 210 30850 21673 32706 1020 86459 Kutaki 370 4755 4493 35354 11933 56905 Launth salla 1000 42554 134342 0 0 177896 Lohan 10835 0 0 570 0 11405 Lokta 64000 43270 87008 11100 9140 214518 Maha 0 0 Main 0 0 Majitho 25201 30246 218 2651 100 58416 Mashala pat 0 0 0 0 20825 Mushali jara 0 11643 1487 0 0 13130 Nagbeli 1300 5787 35 0 0 7122 Nagkeshar 0 200 0 0 0 200 Nigalo gota 0 358000pcs Nirmashi 0 266 974.5 59.5 0 1300 Nundhiki pat 0 3000 0 0 0 3000 Okhar bokra 0 0 275 0 650 925 Paban 0 500 0 0 0 500 padamchal 100 1656 1297 300 600 3953 Pangra 50 100 0 0 0 150 Pipal pat 400 0 0 0 400 Pipla 0 0 1973.5 290 0 2263.5 Raktachandan 0 174 0 0 0 174 Rasulla 0 0 0 14000 0 14000 Ritha 54200 2589.5 150 98410.5 208388 363738

Rudraksha 1050 2797 0 0 0 3847 Salla ko Simta 100 40459 0 0 0 40559 Satawari 0 765 0 5660 27 6452 Satuwa 0 0 347 0 631 978 Setak chini 0 0 0 2448 2448 Shal dhup 0 280 0 0 0 280 Shikakai 200 2304 6800 0 0 9304 Shila jit 0 0 200 47365.5 0 47565.5 Shrikhanda 0 150 0 0 0 150 Simal khoto 0 850 0 0 0 850 Somlata 0 0 1450 2940.5 1770 6160.5 Sugandha kokila 0 0 0 835 0 835 Sugandhwal 0 1427 1610 20813 10481 34331 Sunpati 300 4860 0 0 0 5160 Tapre 0 200 0 0 0 200 Tej pat 0 0 0 3825 10160 13985 Thingure salla 5400 0 0 0 0 5400 Tigeri 0 0 0 5585 0 5585 Timmur 1870 231 125 358573 493 361292 Tite pati 0 5460 0 8663 1073 15196 Tuki phul 0 876 0 2800 0 3676 Pakhanbhed 0 0 1350 0 2997 4347

Lecture 9 October 10, 2004

Name of the herb IRs per kg at Delhi Oct.1st 04

Amaltashphal 12

Anwala 32 –38

Arjun bokra 8

Ashwagandha 45-65

Babari 11-25

Barro bokra 7

Barro singo 4-5

Banapsha 350-550

Banslochannila 35

Bayabidang 35-50

Bel 20-25

Chirayito

Chobchini 11-140

Dalchini 50-51

Guggul 150-175

Harro [syanu+thulo] 9-11

Harro syanu 35-50

Harro thulo 5

Indrajau chal

Jatamanshi 100-130

Jethimadhu

Jhyau 60

Kakoli

Kalmegha 14

Kankarasighi

Kaphal

Kapurkachari 11

Kulanjan

Kutaki 175-210

Laha [pipal] 80-100

Lodha 16-22

Lokhandi 200-225

Maida lakari 20-28

Makoya 35-36

Manjuphal 56-58

Menhandi 30-32

Mushali kalo 50

Mushali seto 250-1350

Nagermotha 9-11

Padamchal 60

Panchaunle 700-1500

Pashanbhed 15

Pipala syanu 90

Pipalathulo 200

Rasot/chutro sar 70-100

Ratanjot

Ritha 15

Salabdana

Salabgantha 65-80

Salabmishri 2800-3200

Sarpagnadha 95-105

Shahijan gunda 35-110

Shatawari /kurilo 110-220

[panhelo]

Shikakai 30

Shilajeet rock/ 150

Shitalchini 192-200

Suddha shilajit 550-1100

Sugandhabal 65

Sugandhakokila 90

Talishpatra 25

Tejpat 25

Timmur 120 Sources of price information: Primary source are the traders dealing the item; and secondary sources are price bulletin like daily and weekly magazines ex Navbharat times; Financial express Alternatively, specialty reporters like Public ledger are the sources of information for prices. Shorting of MAP by volume TOP 10 is

Name Rate(NRs.) Chiaraito 169361.8 200 33872360 Jatamanshi 83590 150 12538500 Jhyau 232677.5 32.5 7562018.75 Kurilo 86459 210 18156390 Kutaki 56905 110 6259550 Lautha Salla 177896 85 15121160 Lokta 214518 100 21451800 Ritha 363738 13.5 4910463 Sila jit 47565.5 150 7134825 Timmur 361292 80 28903360 TOTAL 15,59,10,426.8

Export of NTFPs and their derivatives:

Commodity Revenue Million NRs HERBS 94 Herbal oil 44.5 Herbal Medicine 8.18 Taxus resin 25

Pine Resin 361.6 533.28

� Home assignment: Short out next twenty major plants collected: Give assign Scientific names to the major twenty plants collected. Lecture 10

Challenges in commercialization of plant resources: Let me explain you the case with an instance by a clip from to-days Kantipur: [October 31st, 2004]

Translation by Author: Kantipur reporter: - Sarlalhi, 14 Kartik – A couple dies by eating an herbal drug being sold by himself as a remedy of gastric. Indra Bahdur shrestha and his wife man maya shrestha of karmaiya VDC ward no 1 died by eating an herbal drug called Nirmashi. According to family sources the couple ate 3 pieces of the drug before bed.When the trouble increased they were brought to Ekta Nursing Home.Man Maya was declared dead on arrival to the hospital.Indra Bhadur was refered to Birgunj Hospital the he doed. According to Govind bahadur mainali a local health worker the drug taken was Bish [poison].Indra bahadur used to sale the same drug as nirmashi.According to Deepak Rana Bhat the took the drug clear the bowel toxicity.

The above instance explains how our knowledge on identity & the activity of Herbal crude drug of regarding is fatal to us: Similar knowledge has caused near death of Herbal trade.

1. Local Name: Bikh, Bish

English Name: Nepali aconite Scientific Name: Aconitum spicatum Summary of Information Distribution: Found around 3500m in high altitude. The plant: A herb with tuberous roots, 60-90cm in height. Parts used: tubers, roots Uses: Tubers and roots are used for medicinal

Local Name: Nirbisi, Nirmansi, Nilo bish English Name: Scientific Name: Delphinium denudatum Summary of Information Distribution: which is distributed in between 1300-4500m. The plant: A glabrous branching annual herb, 60-90 cm in height. Found on private lands. Harvesting time during September-October. Part used: roots Properties and uses: The roots are acrid, bitter, thermogenic, digestive, and carminative and are useful in jaundice, fever, ulcer, leprosy, cough and ulcers.

First barrier to commercialization therefore is true identity of the material. Majority herbs are traded in crude form. And the exact identity; and the quality requirements for the products are not defined. WHO and Crude drugs trade:

The plant material, crude drugs are used through out the developed and developing countries as home remedies, over the counter drug products and raw material for the pharmaceutical industries and represent a substantial proportion of the global market. It is therefore essential to establish internationally recognized monographs and analytical guidelines. The World health Assembly –in its resolution WHA 31.33[1978], WHA 40.33[1987] and WHA 42.43[1989 has emphasized the need to ensure the quality of medicinal plant products by using modern quality control techniques and applying suitable standards. International pharmacopoeia provides information on selected medicinal plants that are included in model list, besides many countries has their herbal pharmacopoeia. The WHO monographs are available for a number of globally accepted plant materials though while monographs are not available for regionally traded medicinal plant materials. These pharmacopoeias and monograph have testing procedures for the test of medicinal plant materials. The testing and set standards are optional in Nepal but are mandatory for once the country becomes parties of WTO after 2006. There are ample example of technical barrier that barred Chinese Honey and crude drugs not complying the standards for entry in European market. The following list of test is enlisted in “quality control methods for Medicinal plants”.

1. Determination of foreign matterMacroscopic and microscopic 3. Thin layer chromatography 4. Determination of ash value 5. Determination of extractable matter 6. Determination of water and volatile matter 7. Determination of Volatile oil 8. Determination of bitterness value 9. Determination of hemolytic activity 10. Determination of tannin 11. Determination of swelling index 12. Determination of foaming index 13. Determination of Pesticides residue 14. Determination of arsenic and heavy metals 15. Determination of microorganism 16. Determination of radioactive contamination.

Standards for Spices and Condiments: International Standard Organization [ISO] has 191 technical committees out of which ISO /TC 34 is one. ISO /TC 34 has 15 sub committee one of which is ISO /TC 34 /SC 7. The ISO /TC 34 /SC 7deals with spices and condiments and its secretariat is in Bureau of Indian Standards. The ISO /TC 34 /SC 7 has finalized over 59 international standards for spices. These standards have included testing procedures also. American Spice Traders Association [ASTA] has cleanliness specification of 29 spices.

ASTA specification effective from 1 January 1990 has minimum permissible limit of Whole insect, excreta of mammalian by mg /lb; excreta other by mg/lb; mould % weight; insect defiled % weight and extraneous foreign matter % weight. Nepal Standards of common spices : Part 50,Number 42 of Nepal Gazette section 3 date 2057/10/23 describes standards for Nepal Cardamom [Amomum subulatum], fruit, seed, powder; Dried Ginger, Dried ginger powder; Turmeric, Turmeric powder; Cumin, cumin powder; Whole pepper, pepper powder; Chilies, Chilies powder; Coriander and coriander powder. Nepal Standards of common spices has defined specification based on moisture %, Volatile oil %, Cold-water soluble extract, alcohol soluble extract Total ash Dil HCl insoluble ash and fiber content in some cases. It does refer CaO in dry ginger, lead chromate and curcumin in case of turmeric and fiber content in case of pepper.

Part 50; Number 42 of Nepal Gazette section 3; date 2057/10/23 is silent in many other requirements required globally. It also does not talk on testing procedure. Similarly it is also silent on Zanthoxylum .

Thus, we can infer that quality management and certification is one of the major

Lecture 11 Date: Friday November 05,2004 Section: An Introduction To Natural Product Chemistry: Primary metabolites: Carbohydrates, Proteins, Fats & oils. These primary metabolites in plant chemistry is neither less important nor unrelated to metabolites but because we have limited time in a semester we do not focus here, and also because trade of Jaributi of Nepal [medicinal plant / NTFPs] are often due to unique secondary metabolites in it. Say for example: Asparagus racemosus and Saussurea sps are traded because of unique combination of Polysccharides with glycosides, and traces of alkaloids and other secondary metabolites in it. Secondary Metabolites: Alkaloids, Terpenoids, Carotenoids, Anthocyanins, Flavonoids, Xanthones, and Steroids Here from we enter to organic chemistry

ALKALOIDS: Introduction These compounds are renown for their potent pharmacological activities. Whilst tiny amounts of some can immobilise an elephant or a rhinoceros, others have important clinical use such as analgesics, antimalerial, antispasmotics, for pupil dilation, and treatment of hypertension, mental disorders and tumours. They are all nitrogen heterocycles which occur mainly in plants as their salts of common carboxylic acids such as citric, lactic, oxalic, acetic, malic and tartaric acids as well as fumaric, benzoic, aconitic and veratric acids. Their amine character produce an alkaline solution in water and hence the origin of their name -alkaloids. The alkaloids are extracted from plants by extraction with organic solvent. For example Just as seeds, which are rich in oils, can be extracted with petroleum ether. Solvents like Ethanol and Methanol is also used to extract the alkaloids and after filtration and evaporation the extract is diluted with water acidified and steam distilled to remove the last traces of methanol. After several days at 0 - 5oC the aqueous residue usually clears and it is then possible to separate the organic layer. The organic material is extracted with ether or chloroform, the extracts evaporated and steam distilled again. Volatile alkaloids are separated. The aqueous residue is made alkaline and extracted yet again. The extracts are evaporated and then begin the task of trying to separate the constituents in crystalline form either as the freebase or if not as acid salts. There is a wide variety of structural types of alkaloid e.g. monocyclic, dicyclic, tricyclic, tetracyclic etc. as well as cage structures. Monocyclic alkaloids:

Coniine (1), whose structure is based on piperidine, is highly toxic. It may be extracted from Conium Maculatum [hemlock] and it was used by the ancient Greeks for state executions, Socrates being the most famous victim. On the other hand, nicotine (2), the main alkaloid constituent of tobacco Nicotiana tabacum , is based on the five membered pyrrolidine and six membered pyridine structures. It is without doubt the most well known alkaloid, and its calming effect together with its addictiveness has probably caused the death of more people in the world than any other compound.

The Biosynthesis of Coniine. - Lysine was thought to be the most likely precursor and indeed feeding the hemlock plant with 14C labelled lysine produced radioactive coniine. However it

is possible that lysine is degraded to a simpler compound which is the real precursor. Indeed feeding the hemlock plant with diamino pentane and also with labelled acetate also led to incorporation of the label. The feeding of labelled acetate produces coniine with alternating carbon atoms (2, 4, 6, and 2’) labelled. The high level of label incorporation indicates that the actual biosynthesis of coniine originates from a polyketide. 2. BICYCLIC ALKALOIDS: The tropane alakaloids are based on 1,4 nitrogen bridged cyclohepatane structure.The compound atropine is isolated from Atropa belladonna .

Atropine is widely used in medicine in doses of about 0.1 mg for its muscle relaxant properties. Thus it is used as an antispasmotic including the dilation of the pupil by relaxing the eye muscles and so assists eye treatment, and it is available for the treatment of organophosphate/nerve gas poisoning. Not surprisingly cocaine, which comes from the coca plant, has similar properties to atropine and at one time it was used as a local anaesthetic but is rarely used medically nowadays due to its toxic and addictive effects. There are a number of alkaloids, which are derivatives of quinoline, isoquinoline and their hydrogenated analogues.

Papaverine, an opium constituent, has antispasmotic properties and has also been used as an analgesic. Today it is used as a minor constituent with morphine usually to enhance the analgesic properties of a weaker drug such as aspirin. Emetine is a derivative of tetrahydro isoquinoline which is isolated from the root of a S. African creeper. It has been used as an expectorant, but now replaced by codeine and other non alkaloid drugs such as ephedrine and diphenylhydramine. The most widely used of the quinoline alkaloids is quinine which is isolated from the bark of the cinchona tree. It is used as an antimalarial drug in 0.6 g doses, as a skeletal muscle relaxant it is used in .2 g doses to relieve nocturnal cramps and at trace levels as a bitter flavouring in tonic water. 3. POLYCYCLIC ALKALOIDS The indole structure is also a common feature of alkaloid structures and can be identified as part of polycyclic alkaloids such as reserpine Rauwlofia serpentina , vinblastine of Vinca rosea , strychnine of Strychnos nuxvomica and lysergic acid.

Of these reserpine has the most important clinical use i.e. for the treatment of high blood pressure and as a tranquilliser. Vinoblastine and its analogues are used to treat acute leukaemia, lymphomas and some solid breast and lung tumours. Strychnine is very poisonous and was once used to control rodents, but it has been replaced by poisons which are less toxic to man. The active ingredient of the ergot fungus which grows on cereal grasses such as rye, is a lysergic acid amide. LSD is the diethylamide derivative (X = NEt2) of lysergic acid has hallucinogenic properties. It has no medical applications, but ergotamine tartrate (a tripeptide derivative) is used to treat acute migraine. Its dihydro derivative is even more powerful. Note that Cannabis (marijuana/Indian hemp), which is a mild hallucinogen, is a pyrone dreivative and not an alkaloid. It is also said to be "habit forming"

Morphine, which constitutes ca 10% of the extract from opium poppies, is one of the most potent alkaloids. It is a very effective pain killer and is used in medicine when pain is absolutely intolerable. On the other hand, its acetyl derivative, heroin, is widely abused because of its short-term production of an overwhelming relaxing well-being feeling. Both are highly addictive and with prolonged use produce very harmful physiological effects on the body.

The most commonly used of this class of opioids in medicine is codeine. It is a minor constituent of opium but is made by the methylation of morphine. It is a fairly good analgesic but causes constipation. Thus about 8 mg is often added to either .4 or .5 g. tablets of aspirin or paracetamol. It is also used as a cough suppressant and as an antidiarrhoeal drug. It must be used with care since it is still addictive although to a lesser extent than

morphine.

Lecture 12

November 07,2004

Terpenes

INTRODUCTION Terpenes are probably the most widespread group of natural products. They may be defined as a group of molecules whose structure is based on a various but definite number of isoprene units (methylbuta-1,3-diene, named hemiterpene, with 5 carbon atoms).

This definition leads to a rational classification of the terpenes depending upon the number of such isoprene (or isopentane) units incorporated in the basic molecular skeleton.

Terpenes Isoprene units

Carbon atoms

1 Monoterpenes 2 10

2 Sesquiterpenes 3 15

3 Diterpenes 4 20

4 Sesterpenes 5 25

5 Triterpenes 6 30

6 Carotenoids 8 40

7 Rubber > 100 > 500

Mono-, sesqui-, di-, and sesterpenes contain the isoprene units linked in a head to tail fashion.

The triterpenes and carotenoids (tetraterpenes) contain two C15 and C20 units respectively linked head to head. Many terpenes are hydrocarbons, but oxygen-containing compounds such as alcohols, aldehydes or ketones are also found. These derivatives are frequently named terpenoids [meaning terpene likes].

While the Mono- and sesquiterpenes are the chief constituents of the essential oils the other terpenes are constituents of balsams, resins, waxes, and rubber.

Isoprenoid units are also found within the framework of other natural molecules. Thus, indole alkaloids, several quinones (vitamin K, E) vitamin formed from �-carotene, phenols, isoprenoid alcohol also known as terpenols or polyprenols) also contain terpenoid fragments. The origin of the ubiquitous isoprene unit and its conversion into various compound has been extensively studied. HISTORY Terpenes history spans various civilizations

Ravan [Treta Yug] Arkprakash satak 11th century The Arabs introduced

camphor in Europe from the East.

Arnaud de Villanosa [12th century]

described distillation of oils from rosemary and sage. "oleum mirabile".

Nuremberg [1592] Edited "Dispensatorium valerii cordi"

JJ Houston de la Billardière[1818]

Analysis of oils of turpentine

Dumas [1866] Determined & proposed the name terpene

Wallach [1887] “Isoprene rule” proposed to distinguish the monoterpenes and the sesquiterpenes

Bredt in 1893 Structure of camphor established

Wagner [1894] pinene Tiemann [1895] citral Wackenrodder [1837] �-carotene was isolated

from carrots Willstätter [1907] Correct molecular form ß-

carotene determined

The period since 1945 has seen an extensive explosion in natural product chemistry due to the advent of chromatographic and spectroscopic techniques. Mevalonic acid was shown in 1956 to be a biosynthetic precursor of cholesterol and later, its incorporation into a number of terpenoids has been demonstrated.

Actually, an increasing number of terpenoids are described in the plant kingdom and many of them were shown to have important biological activities. Thus, several sesquiterpenes and diterpenes have antibiotic properties; some sesquiterpenes and diterpenes are insect and plant hormones, respectively.

Componds like Car-3-ene, is present in turpentine oil from pinus, Sabinene is present in Dhupi the Juniper, α- Thujone is presnt in Thuja occidentalis .

Similarly camphor is present in Kapur [Cinnamomum camphora], umbelol in coriander, α-pinene in pine.

SESQUITERPENES

Sesquiterpenoids are defined as the group of 15 carbon compounds derived by the assembly of 3 isoprenoid units and they are found mainly in higher plants. Sesquiterpene structures present several acyclic, mono-, bi-, tri-, and tetracyclic systems. Some of natural sesquiterpenoids are shown below.

The farnesol is present in large quantity in chamomile distilled in Nepal and east Indian sandal oil of Australia. Nerolidol is present on Neroli oil a ctrus type oil.

Similarirly curcumene is yellow pigment in turmeric, Bergamotene is present in “kalo Jyamir” ctrus bergamia

Gossypol is present in cotton seed and Thujopsene in thuja, Cedrol is principle phenol in cedrus deodara [deodar]

DITERPENES

They have 20 carbon atoms and are derived from geranylgraniol pyrophosphate. They are of fungal or plant origin and include the resin acids and the gibberellin plant growth hormones. The diterpenes have exceptionally open chain, as found in geranylgeraniol or phytol, which forms a part of chlorophyll and the side chain of vitamin E and K. Examples of diterpene substances are given below:

Lecture 13 November 19,2004 Carotenes: Yellow or orange-red fat soluble pigments in plants. Distribution in nature: They are widely distributed in nature some common sources of carotenes are Capsicum annum [Chilly], Lycopersicum esculentum [Tomato],Cucurbita maxima [Sweet guard] Carica papaya, Citrus fruits like orange, lemon, mandarins , etc Tomatoes and tomato-based food products are the major source of lycopene and a number of other carotenoids, such as phytoene, phytofluene, -carotene, -carotene, ß-carotene, and neurosporene Other commonly consumed fruits that contain lycopene are pink grapefruit and papaya. Apricots (fresh, canned, dried) also contain low concentrations of lycopene and related carotenoids. Among these foods that are the major source of hydrocarbon carotenoids, only ß-carotene, -carotene, and -carotene are precursors of vitamin A (Fig.). Because the reduced risk of prostate cancer has been specifically correlated with the high consumption of tomato-based food products, this protective effect has been largely attributed to lycopene.

Although lycopene is the major carotenoid in these foods, the presence of a wide range of other carotenoids in tomato-based food products cannot be overlooked.

Uses: It is quite likely that lycopene in combination with other related tomato carotenoids mentioned above might be responsible for the observed biological

activity. In 1995, Tonucci et al. reported on the qualitative and quantitative distribution of carotenoids in name-brand and store-brand tomato-based food products purchased in three major U.S. cities. Analysis: These foods were extracted and analyzed by high-performance

liquid chromatography (HPLC) according to the methodology developed by Khachik et al. other lycopene-containing foods (pink grapefruit, papaya, apricot) as well as those with similar carotenoid profiles (oranges, mandarin oranges, squash).

The carotenoids in these foods have been recently analyzed and quantified by HPLC by the author (F.K.). Although lycopene has, to some extent, been investigated for its biological properties in the prevention of carcinogenesis, Other major hydrocarbon carotenoids have not received much attention.

Therefore, the contribution of other related tomato carotenoids besides lycopene to the chemoprevention of cancer remains unclear.

Lutein

You can see how these and many other carotenes are made up of repetition of isoprene structures.

Question first term November 21,2004

Long question 3x10 -2

1. How many different types of climate are available in Nepal and explain the reason for variation of climate. 2. Validate the statement “Climate has strong effect on vegetation of a location” enlist at list [scientific names] of three plants from growing in each climate zone. 3. Explain, “Knowledge and skill are primary barriers in commercialization of medicinal plants”.

Short questions

1. What do you mean by CBD? Explain articles 15 of CBD. 2. What do you mean by CITES? Explain with example The ban list of Ministry of Forest and soil conservation do not truly reflect CITES spirits! 3. What are alkaloids? Draw the structure of one monocyclic and one bicyclic alkaloid. 4. What are terpenes? Draw the structure α and β pinene 5. What are carotenes? Draw the structures of β carotene.

Very short questions 8 X 2

1. Flora 2. CBD 3. CITES 4. Secondary metabolites 5. Species level diversity 6. Terpenes 7. Alkaloids 8. Carotenes

=========50 marks

Lecture 14 December 3, 2004

Anthocyanins, Flavonoids

Definition,Chemical structure,Distribution in Plants of Nepal, major link with trade

Anthocyanins are naturally occurring compounds that impart color to fruit, vegetables, and plants. Derived from two Greek words meaning plant and blue, Anthocyanins are the pigments that make Vaccinium [ blueberries] blue, raspberries red, Rubus [Aiselu] yellow and are thought to play a major role in the high antioxidant activity levels observed in red and blue fruits and vegetables. Anthocyanins are also largely responsible for the red coloring of buds and young shoots and the purple and purple-red colors of autumn leaves. Close to 300 Anthocyanins have been discovered.

Each fruit and vegetable has its own anthocyanin profile, providing a distinct "fingerprint." Red wine, for example, contains over 15 anthocyanin monomers (type of chemical compound), the varying proportions of which, depending on the type of grape, establish the various shades of the wine's color.

Chemically, they are water soluble flavonoid derivatives, which can be glycosylated and acylated.structures responsible are pelargonidin [scarlet color],cyanidin[ crimson],delphinidin [mauve] color

Numbering in anthocyanins

The aglycone is referred to as an anthocyanidin. There are 6 commonly occurring anthocyanidin structures. However, anthocyanidins are rarely found in plants -

rather they are almost always found as the more stable glycosylated derivatives, referred to as anthocyanins.

Pelargonidin Cyanidin

Delphinidin Peonidin

Petunidin Malvidin

These colors and their combination imparts various color to vegetables and fruits

These colors are PH sensitive

Recently, there has been interest in anthocyanins, not only for their colour properties,

but due to their activity as antioxidants.

Flavones are a group of multi-ring, hydroxyl-containing compounds that are being studied widely for their nutritional value and their use in preventive health care measures. These compounds are found in products as diverse as Ginkgo Biloba, orange juice, and in garden herbs such as dill, oregano and parsley.

Figure shows the structures of some common flavones. Note the large number of OH groups on the rings.

Q: What are anthocyanins? Draw the structure of pelargonidin, cyanidin, Delphinidin, Peonidin, Petunidin, and malvidin .Why anthocyanins have become commercially important these days?

Q: What are anthocyanins? Draw the basic structure of anthcyanins and number the carbon in ring A and ring B.

1. Swertia chirata

2. Nardostachys jatamansi

3. Zanthoxylum alatum

4. Taxus wallichiana

5. Asparagus racemosus

6. Picrorhiza scrophulariflora

7. Lichen sps

8. Accacia sps

9. Daphne bholua

10. Shilajit

11. Terminalia sps

12. Emblica officinalis

13. Mentha sps

Lecture 15 December 5, 2004 Xanthones, and Steroids Xanthone are ketone with a molecular formula C13H8O2 that is the parent of several natural yellow pigments.

xanthone skelton & numbering

The molecules as stated above are present in several medicinal plants and are medicinally much important. These 1,2,3,4,5,6,7,8 carbons can be substituted by hydroxyl and methoxy function, which give various compounds. Xanthone derivatives are present in several plants traded from Nepal one such plant is Swertia chirata : Which contains 1,8-DIHYDROXY-3,7-DIMETHOXYXANTHONE antitubercular Swerchirin: 1,8-dihydroxy-3,5-dimethoxyxanthone . Hypoglycemic xanthones are present in several other members of gentian family like Picrorhiza kurroa Andrographis paniculata is yet another plant with xanthone.It has following four

xanthones (i) 1,8-di-hydroxy-3,7-dimethoxy-xanthone, (ii) 4,8-dihydroxy-2,7-dimethoxy-xanthone, (iii)1,2-dihydroxy-6,8-dimethoxy-xanthone and (iv) 3,7,8-trimethoxy-1-hydroxy xanthone Garcinia cambogia, rheediaxanthone A ,garbogiol, was isolated from the root;

Mangostein is another fruit with high xanthones, which are commercially important. Like anthocyanins, which we discussed earlier, they also have free radical scavenging property and have become extremely important in recent years.

Steroids

Steroids are fat-soluble hormones with a tetracyclic base structure. The base structure consists of four fused rings: three cyclohexane rings and one cyclopentane. The basic structural backbone can be seen below:

As you can see, each of the rings is designated by a letter. Rings A and D are the most commonly modified rings. The following diagram shows the numbering of the carbons in steroids, which will be useful later in this article. The two methyl groups on C10 and C13 are also designated with numbers, as they are present in most steroids.

Steroids are synthesized in the body from squalene, a complex linear aromatic Molecule.

Steroids have a variety of uses in the human body, including, but not limited to: controlling meiosis, carbohydrate metabolism, fat storage, muscle growth, immune

function and nerve cell membrane chemistry. Steroids can be separated into three main groups: gonadal compounds, glucocorticoids and mineralcorticoids. This distinction depends on the site of synthesis of the steroid. The gonadal variety are mainly synthesized in the gonads, as is suggested by the name, while the glucocorticoids (eg cortisol, cortisone) and mineralcorticoids (eg aldosterone) are synthesized in the adrenal cortex.

Steroids can also be divided into groups by function: androgens, estrogens, progestogens, anabolics, and catabolics. The two main types of steroids that we will consider are anabolics and androgens. Androgens exert some kind of masculinizing physical effect on the body, while anabolics promote growth. However, these distinctions are not completely exclusive. For example, testosterone is synthesized by the adrenal cortex as well as the testes, and has both anabolic and androgenic properties.

Cholesterol well known molecule supposed to be responsible for cardiac troubles. Molecules like Pregnenolone is a hormone secreted in the uterus controlling ovum implantation, and is the precursor for the androgens, estrogens, and glucocorticoids.;Testosterone are synthesized in testes

Steroidal skeletons are present in several plants of Nepal:

Several plant traded from Nepal are primarily traded because one or the other

steroid is present in it. Dioscorea genera is source of Diosgenin which can be converted Pregnane and several hormonal drugs Asparagus spp phytoecdysteroids Cordyceps sinessis antitumor compounds 5 ,8 -epidioxy-24(R)-

methylcholesta-6,22-dien-3 - -glucopyranoside and 5,6-epoxy-24(R)-

methylcholesta-7,22-dien-3 -ol Dactylorhiza latagirea ecdysteroids Dioscoria spp. diosgenin Panax ginsenoside-Rh1, a component of ginseng saponin, activates estrogen receptor in human breast carcinoma MCF-7 cells Sapindus mukorossi mukorossides industrial saponin for production shampoos and fiber softening agent. Saussurea costus : Four steroids were isolated from the Saussurea gossypiphora for the fist time. They were determined as 3-stigmastanol, beta-sitosterol, stigmast-7-en-3-ol and ergostan-3,24-diol by spectral and chemical methods. Paris polyphylla : phytoecdysteroids

Lecture 16 December 12, 2004

Name kg Chiaraito 169361.8 Jatamanshi 83590 Jhyau 232677.5 Kurilo 86459 Kutaki 56905 Lautha Salla 177896 Lokta 214518 Ritha 363738 Sila jit 47565.5 Timmur 361292 TOTAL

CHIRATA Scientific Name: Swertia chirata Buch.-Ham.ex Wall. Cat. n. 4372. Original Data Notes: Reg. Himal English: Chirata Local name : Tite,Tikto,Chirayito,Chirayata ,Chirata Family : Gentianaceae

Genus swertia is represented by over 21 species in Nepal out of which about 9 species appear in trade. S.alata, S.anguistifolia,S.bimaculata,S.Chirata,S.multicaulis,S.nervosa,S.paniculata,S.purpurascens,S.racemosa among those in trade. Trade: 055/056 056/057 057/058 058/059 059/060 166451

169361

337497.5 Missing 119315.5

Average flow per year 224436 kg per year Royalty Rate NRs 3 per kg The price trend

NRs 20/- per kg in 2040 NRs 120/- per kg in 2051; NRs 500 per kg in 2058;

Chemistry

Gentianine

Pharmacology UNIQUE CHEMICALS OF SWERTIA CHIRATA Amarogentin

1,8-DIHYDROXY-3,7-DIMETHOXYXANTHONE Plant: Antitubercular; Pesticide BETA-AMYRIN Plant: Antiedemic IC27=40 mg/kg ipr rat; Antiinflammatory; Antinociceptive GENTIANINE Plant: Analgesic; Antianaphylactic; Antiarthritic; Antibacterial; Antiedemic; Antihistaminic; Antiinflammatory; Antimeningitic ivn man; Antipsychotic; Antirheumatic; Antishigellic; Antistaphylococcic; Antistreptococcic; Ataractic; CNS-Paralytic; CNS-Stimulant; Corticosterogenic; Emetic; Hypoglycemic; Hypotensive; Myorelaxant; Neurotoxic; Pesticide; Sedative GENTIOPICRIN Plant: Antimalarial; Fungicide; Larvicide 1,300-2,00 mg/man; Pesticide LUPEOL Plant: Antiedemic; Antiflu; Antihyperglycemic; Antiinflammatory 1/3 Indomethacin; Antilithic 25 mg/kg/day; Antimalarial IC50=46.8 ug/ml; Antioxalate 25 mg/kg/day; Antioxidant; Antiperoxidant; Antirheumatic; Antitumor; Antiurethrotic; Antiviral; Cytotoxic 50-500 ppm; FPTase-Inhibitor IC50=65 ug/ml; Hypotensive; Pesticide; TOPO-2-Inhibitor IC50=10.4 uM OLEANOLIC ACID: plant SWERCHIRIN Plant: Antihepatotoxic; Hypoglycemic; MAO-Inhibitor SWERTIANIN Plant: Antitubercular; Mutagenic; Pesticide CHEMICALS COMMON TO OTHER PLANT GENERA MANGIFERIN Plant: Antidermatitic; Antidiabetic; Antifibrotic; Antihepatotic; Antihepatotoxic; Antiherpetic; Antiinflammatory; Antioxidant; Antispasmodic; Antiviral; Cardiotonic; Choleretic 20 mg/kg; CNS-Stimulant 50-100 mg/kg; Diuretic; Immunostimulant; MAO-Inhibitor; Pesticide; T-Cell-Stimulant TANNIN Plant: Anthelmintic; Antibacterial; Anticancer; Anticariogenic; Antidiarrheic; Antidysenteric; Antihepatotoxic; AntiHIV; Antihypertensive; Antilipolytic; Antimutagenic; Antinephritic; Antiophidic; Antioxidant 1/3 quercetin IC50=1.44 ug/ml; Antiradicular 1/3 quercetin 500 mg/kg/day orl mus; Antirenitic; Antitumor; Antitumor-Promoter; Antiulcer; Antiviral; Cancer-Preventive; Carcinogenic; Chelator; Cyclooxygenase-Inhibitor; Glucosyl-

Transferase-Inhibitor; Hepatoprotective; Immunosuppressant; Lipoxygenase-Inhibitor; MAO-Inhibitor; Ornithine-Decarboxylase-Inhibitor; Pesticide; Psychotropic; Xanthine-Oxidase-Inhibitor OLEIC-ACID Plant: 5-Alpha-Reductase-Inhibitor; Allergenic; Alpha-Reductase-Inhibitor; Anemiagenic; Antialopecic; Antiandrogenic; Antiinflammatory IC50=21 uM; Antileukotriene-D4; Cancer-Preventive; Choleretic 5 ml/man; Dermatitigenic; FLavor FEMA 1-30; Hypocholesterolemic; Insectifuge; Irritant; Percutaneostimulant; Perfumery; Propecic PALMITIC-ACID Plant: 5-Alpha-Reductase-Inhibitor; Antialopecic; Antiandrogenic; Antifibrinolytic; Antioxidant IC40=60; FLavor FEMA 1; Hemolytic; Hypercholesterolemic; Lubricant; Nematicide; Pesticide; Propecic; Soap STEARIC-ACID Plant: 5-Alpha-Reductase-Inhibitor; Cosmetic; FLavor FEMA 2-4,000; Hypocholesterolemic; Lubricant; Perfumery; Propecic; Suppository

NO ACTIVITY REPORTED SWERTININ, OPHELIC-ACID CHIRATIN Plant:

ppm = parts per million tr = trace

Lecture 17

December 17, 2004

Essential oils EXPORT Nepal exports under HS code 3301[3301 Essential oils; resinoids; terpenic by-products etc]; 55 ton worth 335000 US$, representing 2% of global trade positioning the country 72 in the list of exporters of the category: Other major players are ranked below

[HS code 3301] Year 2003

US $ thousand Quantity in Ton % share

World estimation 1,606,725 196,492 100 United States 282,010 27,944 17

France 191,905 6,056 11 Brazil 114,385 69,521 7

United kingdom 108,575 6,856 6

India 101,987 8,778 6 China 62,873 10454 3

Argentina 56,019 4,977 3

These five-countries account for 53 % of total essential oil traded in the world. Nepal’s export of essential oil under HS code 330129 [essential oils, whether or not terpeneless, incl. concretes and absolutes (excl. those of citrus fruit, geranium, jasmine, lavender, lavandine, mint and vetiver)] Nepal exported about 34 ton of essential oil under HS code worth 389000 US$ of which 29 ton (60%) of essential oils were exported to India. Other countries to which essential oil under HS code 330129 were exported are Belgium, Austria, Hungary, Spain, and Germany t. This comprised mainly of grass oils. Nepal exported mainly

1. Acorus calamus oil. [af f]emf] t]n ]

2. Artemisia vulagaris oil.[ tLt]kftL t]n]

3. Cinnamomum glausecens oil [ ;"uGw sf]sLnff t]n ]

4. Curcuma zeodaria oil [sr"/ t]n ]

5. Cymbopogon flexuosus oil [n]dgu| f; t]n].

6. Cymbopogon martini [kfdf/f]h f t]n].

7. Cymbopogon winterianus oil [;L 6| f] g]n f t]n]

8. Gaultheria fragarantissima oil[ w ;L=u|] t]n]

9. Juniperous recurva oil [w"kL t]n ]

10. Matricaria chamomilla oil.[s]df]dLn t]n ]

11. Mentha arvensis oil.[d]+yft]n]

12. Nardostachys grandiflora oil[h6 fd;L t]n].

13. Ocimum basilicum oil[ a] ;L n t]n ].

14. Rhododendron anthopogon oil.[;"g k f tL t]n ]

15. Zanthoxylum armatum oil[ 6 L Dd" / t]n ]

16. Turpentine oil.*[ tf / kL g t]n ] IMPORT

HS Code Essential Oil imported Quantity ton

Cost US$

Supplier

3301 Essential oils; resinoids; terpenic by-products etc

515000 Ind,Sing,Uk

330124 Mentha piperita oil 23 145000 India 330125 Mentha arvensis oil 4 23000 India 330129 Essential oils, whether or not

terpeneless, incl. concretes and absolutes (excl. those of citrus fruit, geranium, jasmine, lavender,

1 13000 India

lavandine, mint and vetiver) Total import in US $ 696000

JATAMANSI [updated on December 17, 2004] Family : Valerianaceae All the classical information are recorded by the name Valerianaceae Nardostachys DC. Coll. Mem. vii. 4. tt. 1, 2 (1830).

Original Data

Notes: Benth. & Hook. f. Gen. Pl. ii. 153. {Corrected when editing for database} The original plant of CIS-Himalayas perhaps was 1. Nardostachys jatamansi DC. Coll. Mem. vii. 4. t. 2 (1830). Original Data Notes: Reg. Himal 2. Nardostachys grandiflora DC. Coll. Mem. vii. 4. t. 2 (1830).

A Collector with N. grandiflora

Original Data Notes: =Nardostachys Jatamansi The species was also recorded by earlier Botanist at nearly similar altitude but with bigger leaf and prominent flower

3. Nardostachys gracilis Kitamura in Acta Phytotax. & Geobot., Kyoto, xv. 134 (1954). This species is recorded from Manang /Nepal Nardostachys chinensis Batalin in Act. Hort. Petrop. xiii. (1894) 376. The species may occur in trans Himalayas may be the plant recorded by Kitamura from Manamg is N.chinenesis All 3 species N.jatamansi, N.grandiflora and N.gracilis are treated as synonym by current botanist. Perhaps the confusion came after 1960 when the taxonomist using the morphology as key for classification found similar morphology. Distribution: commercially the “Jatamansi” is collected from Taplejung and Terhathum in eastern Nepal.Dolkha,Rasuwa in central Nepal Manang and Gorkha in western Nepal Humla,Jumla,Dolpa,Mugub and Bajhang in mid western Nepal and Darchula in far western Nepal. Though the plant species is present in all mountain districts. HISTORY OF USE The plant is in human use since vedic era / Charak samhita also describes the use of jatamansi. “The plant is in religious, and cultural use and is also mentioned in “Durga Saptasati” where is said to used “Hawan”. The plant is in found to used by ethnic groups of Jordan, turkey and Israel and mentioned in Bibilical citation where the medicinal property has been attributed. TRADE Perhaps this wide application has resulted the trade of the plant since olden days and appears in trade by various name English : Spikenard Nepali : Jatamansi,Jatamasi, Humli and people in western Nepal call it Bhulte In Indian market it is called “Jatamansi & Balchar” LEGAL STATUS Nardostachys was added to the Convention on International Trade in Endangered Species (CITES) in 18.09.1997 at the request of India. Only the bulk herb materials (whole and sliced roots) are currently restricted from trade; manufactured preparations, such as powders, pills, extracts, and teas are still permitted in trade. Neighboring Nepal also recognized the declining supplies of the herb, stating: The HMG Nepal (1991) formally recognizes the need to preserve the threaten species restricts trade except in unprocessed form . Jatamansi (Nardostachys grandiflora) is banned trade in crude form. However large quantity of crude drug is still collected and traded both in processed and un processed form. TRADE VOLUME:

055/056 056/057 057/058 058/059 059/060 29068 83210 51936 46734

In average 54738 kg per year Jatamanshi has collected and traded in past three years. The royalty rate per kg is NRs 25 and NRs 13,686,50/- has been collected per year The average price per kg of jatamanshi rate was found to be above 120 /- and thus NRs 65,68,560/- worth of Jatamanshi appeared in trade in these years. The price hike in last decade is NRs 65/- per kg NRs 165/- per kg.

Olsen (1999) estimated that Nepal’s annual export of dried unprocessed rhizomes of Nardostachys grandiflora is approximately 1000 ton per year.Bhattarai(2000) “marc” of N.grandiflora exported from Nepal to India was 3202 kg throough

CHEMISTRY :

EO Root 19,000 - 20,000 ppm

1(10)-ARISTOLEN-2-ONE Root 18 - 120 ppm

18,9,10-DEHYDROARISTOLAN0NE-(2) Root 6 - 40 ppm

ALPHA-PATCHOULENE Root:

BETA-IONONE Root 25 - 180 ppm

BETA-PATCHOULENE Root:

ISOVALERIC-ACID Root:

JATAMANSIC-ACID Root:

JATAMANSIN Root:

JATAMANSINOL Root:

JATAMANSONE Root:

MYRISTIC-ACID Plant:

N-HEXACOSANE Root:

N-HEXACOSANOL Root:

N-HEXACOSANYL-ISOVALERATE Root:

NARDOL Root:

NARDOSTECHONE Root:

NORSEYCHELANONE Root:

PATCHOULI-ALCOHOL Root:

VALERANONE Root:

Two guaiane- and two aristolane-type sesquiterpenoids (1–4) were isolated from Nardostachys chinensis roots, and none exhibited antimalarial properties.

PHARMACOLOGY Salim S et al (2003) have shown the protective effect of Nardostachys jatamansi (NJ) on neurobehavioral activities, cerebral ischemia in rats. The study provides first evidence of effectiveness of NJ in focal ischemia most probably by virtue of its antioxidant property. The known sesquiterpene valeranone (= Yatamanson) was isolated from the subterranian parts of Nardostachys yatamansi (DC). It was pharmacologically investigated in animal experiments of sedative, tranquilizing and antihypertensive properties A weak activity hypotensive was demonstrated on ratsand mice . The toxicological studies on rats and mice showed an oral LD50 of greater than 3160 mg/kg was found, which suggests the possibility of a therapeutically useful dose ratio. In three other pharmacological models an anti-ulcer action was detected Ali S et al (2002) has shown that 50% ethanolic extract of the rhizomes of N. jatamansi is shown to possess hepatoprotective activity in rats at (800 mg/kg body wt, orally).

Lecture 18

December 19, 2004

Family : Rutaceae 1. Zanthoxylum armatum DC � Syn :Zanthoxylum alatum Roxb � Syn: Zanthoxylum acanthopodium DC 2. Zanthoxylum oxyphyllum Edgew Trade name Eng: Indian Prickly Ash; Hindi : Tomer beej ; Nepali : Timmur, Bale timur;

Tejmal ; Yerma

Plant habitat: Forest undergrowth and hot valleys. 600 to 2100 metres in the Himalayas. Plant Distribution

The plant is found as an under growth in eastern to western mountains. In the east it is distributed from 600m to 1800 m .Mid western mountain region is the principal region of collection though small amount is collected from eastern and far western- mid hill also. It is found up to 2100m in the western Nepal. It is also found in trans Himalayan Districts and southern china. Plant description

Zanthoxylum is an armed,scandent or esrect shrub or a small tree up to 6m high with dense foliage. The branches are armed with flattened prickles up to 2 cm long. Bark is pale brown ,deep furrowed imparipinate or tri foliate. The leaves are 5 to 25 cm in length often with lanceolate shape, entire to grandular crenate margin ; acute or obtusely acuminate ;Flwoers white to greeninsh in dense terminal or axillary panicles ;Fruit sub globose ,glabrous with shining black seed

The plant prefers light (sandy), medium (loamy) and heavy (clay) soils and requires well-drained soil. The plant can grow all type soil [acid, neutral and basic

Use Older references are found in Charak ( ;' c @ ; tLSt :sGw la c * )

Part used: ripe fruit, steam bark, wood. � Used as spice � The fruit contains 1.5% essential oil.

The fruit is used to purify water. Toothbrushes are made from the branches. Wood - heavy, hard, close-grained. Used for walking sticks.

Trade:

055/056 056/057 057/058 058/059 058/059 355403 361292 553792 NA 650193.8

0

100000

200000

300000

400000

500000

600000

700000

055/056 056/057 057/058 059/060

Series1

Export of Zanthoxylum alatum fruit [timur] from Nepal

The of Price of Zanthoxylum alatum fruit [timur] in 1991 was NRS 20/- per kg and in 2001 the price NRs 85/- per kg the current year price is about 120/- per kg All most entire of timur produced in the country goes to India. Dabur is the principal buyer which now pulverizes in Nepal and exports to Dabur India as “Dantasakti” In the year 2003 Zanthoxylum alatum worth NRs 78,023160/= was traded and NRs 1,968964.30 revenue was generated. Tigeri is the principal aultrant besides fruits of other z.species. Chemistry: Plant part Chemicals Concentration

α -Phellandrene 10,000 to 20,000 ppm

Linalol 14,745 ppm

Linalyl actate

Sabinene

Citral

Fruit /pericarp

Geraniol

Limonene

α –Phellandrene Linalol Linalyl actate

Isoquinoline alkaloids present in the Zanthoxylum alatum bark and roots;

Magnoflorine 1700 ppm

Berberine

Leaf contains linalool and linalyl acetate Seed of zanthoxylum alatum contains fixed oil

Pharmacology

Das et al (1999) have noticed mosquito repellent property zanthoxylum armatum oil. At 0.57 mg/cm2 concentration timur oil in brassica oil gave significantly higher protection up to 445 min.It was found to be better than dimethyl phthalate.

Kumar S and Muller K have noticed antiproliferative activity against the growth of human keratinocytes.

Manandhar N.P. (1995) has documented traditional application of fruit of Zanthoxyllum in abdominal pain.

Joshi K and Joshi A.R.(2000) have recorded the traditional use of this zanthoxylum armatum fruit in gandaki valley.

D.Eigner and D.Scholz (1996) have recorded 05 to 1.5 g of zanthoxylum armatum fruits are used in Nepali cuisine.

Nath DR have compared leech repellent action in volatile oil of Zanthoxylum armatum DC. syn. Z. alatum Roxb (Timur) .According to Nath et al to Timur oil was at par with Citronyl and exhibited better results than dimethyl phthalate (DMP) and N-benzoyl piperidine (NBP).

Xiong QB and Shi DW have recorded its traditional use of pericarp of zanthoxylum fruit “Hua Jiao” in epigastric pain accompanied by cold sensation, vomiting, diarrhea and abdominal pain due to intestinal parasitosis, ascariasis and used externally for eczema in China.

References:

1. J Commun Dis. 1999 Dec; 31(4): 241-5. 2. Phytother Res. 1999 May; 13(3): 214-7. 3. A 4. B 5. Indian J Med Res. 1993 May; 97:128-31. 6. Yao Xue Xue Bao. 1991; 26(12): 938-47.

Class test question

What are anthocyanins ? draw the structure of pelargonidin and cyanidin.

What are xanthones ? How many species of swertia are traded from Nepal Name the unique compounds of Swertia chirata.

What are HS codes for essential oil exported from Nepal? Write the pharmacological effect of any one Nardostacys jatamansi or

Zanthoxylum alatum

Draw the basic skelton of anthocyanins, Flavones, and Xanthones showing numbering as per IUPAC system.

Write short notes on Pahrmacological activity of any one of the followings

anthocynin, (b) flavone,(c) xanthone, (d) terpenoids (e) alkaloids

Lecture 20

December 26, 2004

Sapindus mukorossi [Rittha]

Family :Sapindaceae

Scientific name : Sapindus mukorossi Gaertn. Fruct. i. 342. t. 70.

Sapindus emarginatus Hort.Alger. ex Rev. Hortic. lxvii.

(1895) 303.

Distribution: Asiatic tropic; Tropical and sub tropical Nepal

Description: Deciduous to small tree with big compound leaves ,odd pinnate ,long stalked up to 50 cm long; 8 –13 leaflets oblong lanceolate , opposite or alternate ,entire, bright green ,glabrous,6-18 cm long and 2-4.5 cm wide.Apex acuminate, base obliquely cuneate.Small yellowish flower 3-4 mm across in 10 – 25 cm long terminal panicles;

Flowers in July; fruit globose 2cm across of yellowish –brown color.

Traded part: Ripe yellowish to brown fruits.

Traded Volume: unit kg

055/056 056/057 057/058 058/059 059/060 38376 363738 450381 616333

Royalty rate is NRs 2/-kg . Average price in the year 2040 was 11.NRs per kg in Nepalgunj market and in the year 2061 NRs 17 at Nepalgunj..Thus worth NRs 10477661of Rittha was traded

Part in use: pericarp

Sapindus mukorossi fruit comprises of 50to 55 % pericarp and 45-50% seed.

The pericarp contains 6.1 to 8.4 % of saponin for which the soap nut are traded.

Typical saponin structure

Saponin = Glycone + aglycone

In Sapindus mukorossi nuts principle sugar is glucose molecule and the basic steroidal saponin skelton present is Hederagenin.

Extraction of Saponin:

1. Soap-nut pericarp can be extracted by ethylacetate. The solvent is recovered by vaccum evaporation. The residues are dissolved in water, allowed to settle. The clear supernatant layer is drained. and the aqueous layer thus obtained is treated with Bariumhydroxides. Saponins separate as barium salt of saponins.The barium-saponin salts are suspended in ethylalcohols and carbondioxide is bubbled where by bariumcarbonate precipitates and saponin is left in the solution which can be separated by evaporating the water. 2. Saponin can also be extracted by 50% alcohol and precipitation by ethers. 3. Commercially viable method that has been patented by J.Gedeon

1951 has Indian patent number 46945.

This method uses advantage of solubility of saponin in water.In this method [which you have done in laboratory also] the coarse pericarp is extracted with boiling water until no saponin is left.Hot aqueous extract is cooled and ammonium sulphate is added where by saponin is precipitated.The precipitated saponin is brown in color and can be cleaned by repeated dissolving and precipitation or recrystalizing in appropriate solvents.

Soap nut kernel gives 24 to 35% of fixed oil on solvent extraction .The fatty acid composition of sapindus mukorossi seed oil was determined by spectrophotometery,urea complexation and gas liquid chromatography (GLC).

The percentage of individual fatty acids were found to be Palmitic acid 4.0;stearic acid 0.2;arachidic acid 4.4;oleic acid 62.8;linileic 4.6;linolenic 1.6;eicosenoic acid 22.4.

The Triglyceride composition was determined from native oils and of the monoglycerides produced from it by pancreatic lipase hydrolysis.The oil contains 0.1% trisaturated ;2.1% monosturated and 75.8% triunsaturated acids.

The special characters tics of sapindus mukorossi seed oil is its content of 26.3 and 26.7% triolein and eicoseno-di-oleins respectively [Sengupta A. Basu SP,Saha S; Lipids 1975 January 10:33-40].

Uses: traditionally soap nut is used to clean wool, hair and ornaments.

Industrially it is used soften the hides, and froth floatation of metals from ores and minerals.

Pharmacologically verified application: It is anti-inflammatory and anti fungal.

In the recent year the volume of trade has grown tremendously because of its application in herbal shampoo.

Steroid sources of Nepal

Several plant traded from Nepal are primarily traded because one or the other steroid

is present in it.

Dioscorea genera is source of Diosgenin which can be converted Pregnane and

several hormonal drugs

Asparagus spp phytoecdysteroids

Cordyceps sinessis antitumor compounds 5 ,8 -epidioxy-24(R)-methylcholesta-

6,22-dien-3 - -glucopyranoside and 5,6-epoxy-24(R)-methylcholesta-7,22-dien-3 -

ol

Dactylorhiza latagirea ecdysteroids

Dioscoria spp. diosgenin

Panax ginsenoside-Rh1, a component of ginseng saponin, activates estrogen receptor

in human breast carcinoma MCF-7 cells

Sapindus mukorossi mukorossides industrial saponin for production shampoos and

fiber softening agent.

Saussurea costus : Four steroids were isolated from the Saussurea gossypiphora for

the fist time. They were determined as 3-stigmastanol, beta-sitosterol, stigmast-7-en-

3-ol and ergostan-3,24-diol by spectral and chemical methods.

Paris polyphylla : phytoecdysteroids

Lecture 21

Taxus /taxane/ taxol form original file

Lecture 22

December 30, 2004

Asparagaceae

Asparagus racemosus Willd

Protasparagus racemosus (Willd.) Oberm

Ind. or.; Afr. trop.; Austral

Asparagus adscendens Roxb.

Reg. Himal. Afghan

Local Name: Shatavari, Kurilo,

The genus Asparagus has been recently moved from the subfamily Asparagae in the family Liliaceae to a newly created family Asparagaceae.

Distribution: The plant has almost global distribution. The specimen of Asparagus racemosus has been collected from Africa, Australia, Asia. The plant is found in tropical and and subtropical Nepal.

Major districts of collection are: panchthar,sunsari,terhathum,

DESCRIPTION:

The plant is a climber growing to 1-2 m in length. The leaves are like pine needles, small and uniform. The inflorescence has tiny white flowers, in small spikes. The roots are finger-like, and clustered.

Traded part: finger like of Roots both shatavari and kurilo appear in trade.

Collection and Trade:

055/056 056/057 057/058 058/059 059/060 43825 86459 107659 94756

Average flow per year in the above mentioned year is 83174.75 kg.

Price in the year 040 was NRs 50/- per kg for sahtavari and NRs 30/- per kg for kurilo.This year shatavari sold up to 225 /- per kg in nepalagunj and kurilo was rated up to 175/- per kg .

Chemistry:

Ecdysteroids

Traditionally Asparagus root [shatavari] is used as galactogauge, and vitiating to nourishing mothers.

Asparagus racemosus (Shatavari) is recommended in Ayurvedic texts for prevention and treatment of gastric ulcers, dyspepsia and as a galactogogue. A. racemosus has also been used successfully by some Ayurvedic practitioners for nervous disorders, inflammation, liver diseases and certain infectious diseases.

Pharmacology:

More recently Wiboonpun N. [2004] et al has established its anti oxidant activity due to compounds like racemofuran, asparagamine A and racemosol .

A review paper by Goyal et al (2003) gastrointestinal effect; galactogauge effect [increase milk in pet animals and nourishing mother; Inspite of cholinergic activity of A. racemosus on guinea pig's ileum, ethyl acetate and acetone extracts of the root of A. racemosus blocked spontaneous motility of the vi

Immunomodulating property of A. racemosus has been shown to protect the rat and mice against experimental induced abdominal sepsis rgin rat's uterus.

Alcoholic extract of root of A. racemosus has been shown to significantly reduce the enhanced levels of alanine transaminase, aspartate transaminase and alkaline phosphatase in CC14-induced hepatic damage in rats

Chloroform/methanol (1:1) extract of fresh root of A. racemosus has been reported to reduce

the tumor incidence in female rats.

Alcoholic extract of the root of A. racemosus has been reported to produce positive

ionotropic and chronotropic effect on frog's heart with lower doses and cardiac arrest with

higher doses. The extract was found to produce hypotension in cats

Higher doses of the alcoholic extract of root of A. racemosus are reported to cause dilatory

effect on bronchial musculature of guinea pigs but failed to antagonise the histamine induced

broncho-constriction.

Alcoholic extract of root of A. racemosus was found to have slight diuretic effect in rats and

hypoglycemic effect in rabbits, but, no anticonvulsant and antifertility effect was observed in

rats and rabbits respectively. However, it did show some anti-amoebic effect in rats.

In Ayurveda, A. racemosus has been described as absolutely safe for long term use, even

during pregnancy and lactation. Systemic administration of higher doses of all the extracts

did not produce any abnormality in behaviour pattern of mice and rat

References:-

1. Phytother Res. 2004 Sep; 18(9): 771-3.

2. Goyal RK, Singh J, Lal Harbans Pt. B. D. Sharma Post Graduate Institute of Medical Science, Rohtak, Haryana, India

Lecture 23 Date December 31, 2004 English: Picrorhiza /Kutaki/ Kataki Nepali name: Kutaki /Katuki/katuko Scientific name: Picrorhiza kurroa Royle ex Benth. Scroph Ind 47(1835) Picrorhiza kurroa Royle Illustr.Bot.Himal 291 Picrorhiza scrophulariiflora Pennell. Scroph.W.Himal (Acad.nat.Sci.Philad.,Monograph.5 ) 65(1943) Base Name Neopicrorhiza scrophulariiflora (pennell) D.Y.Hong Original data Notes: Ind.or (Sikkim);China (Yunan) Illus Family: Scrophulariaceae [Gentian family]

Distribution: It flourishes well in the rocks and crevices in the upper grassland with Nardostachys in Alpine himalays 3000 to 5000 meter [Western to eastern himalyas upto Sikkim ] in trans himalyas up to China (yunan)

Plant description: It is a small perennial herb with long creeping root and small flat oval and serrated leaves. Flowers are on long spike that appear June -August. The flower color white to pinkish white.

Lecture 24 Date January 9, 2005 LICHEN SPECIES IN TRADE Lichen is best form of symbiotic life. It is a symbiosis of Algae and fungi. Fungi provide moisture and mineral material and algae, prepare food from atmospheric Carbon dioxides, and elaborate its food to fungus partner.

Local name: Nepali Jhyu Trade name: Charila, Patther ka phul English name: Lichen

Classification: Basis of classification is fungus and its algal associates. When Fungus is

� Ascomycetes � Basidiomycetes

The algal associates are

Myxophyceae (Blue green algae) Chlorophyceae (Bright green algae)

� When thalli is thin crust like it is crustose � When thalli is thin leaflike,flat and leathery it is called Folliose � When thalli is erect simple or branching it is called fruiticose.

Greater part of lichen thallus is composed of fungus hyphae woven together into a compact false tissue. In advanced forms it is divided into Upper cortex of compact hyphae Medula, which is loose, layer hyphae. Gonidial layer of algal tissue Ascolichens are more important commercially and are divided into two series Gymnocarpae with apothecia pyrenocarpae with perithecia

Basidiomycae are less important commercially and distributed in tropics Following families falling in gymnocarpae are economically important and appear in trade.

1. Cladoniaceae 2. Gyrophoraceae 3. Lecanoraceae 4. Parmeliaceae 5. Roccelleceae 6. Peltigeraceae 7. Usniaceae

Evernia prunastri [65/-]

Ramalina farinacea

Cetraria islandica

Usnea barbata

Trade volume from Nepal

055/056 056/057 057/058 058/059 059/060 153519 kg 192285 kg 95557 kg NA 205985.

The price of Lichen in the 048 was NRs 8/- per kg and on December 12,2004 Evernia was selling @ 65/-kg; and for Stone flower minimum price was NRs 75/- per kg. The price rise is 8.75 times which can mainly be attributed to HMG ban on trade of the species. The lichen is ban in trade in unprocessed form; no processors are currently processing lichen because of mixing of species by collectors and traders. However Indian buyers are buying shorting and selling to niche market within India. The increase in trade volume can be further to INGO facilitation of NTFPs trade and familiarity of more and more collectors with this item of trade. HPPCL which processed in its earlier days stopped it because in competency of quality. The trade of crude lichen continues. The average volume traded per year based on available statistics of collection in stated years shown above is 161836.5 kg. Royalty rate per kg is NRs 10/- thus the revenue to the state in these years was about NRs 1618365/- per year and when sold to Indian buyer at minimum price @ 65/- return to trade was not less than 10519372.5/-] and net return to the country is NRs 12137737.5 Chemical composition of Lichen Carbohydrate

1. Lichenin 2. Isolichenin

The carbohydrate soluble in hot water and insoluble in cold water

Protein similar to casein Vitamins like

1. Riboflavin

2. Carotene Others Acids: Usnic and Lecanoric acids. The former is responsible for lots of medicinal property while latter is responsible for dye value of lichen

1. Lecanoric acid + ammonia = orcein ----------- orchil ---------Na, K azolitmin / erythrin ------------/////erythrolitmin

LITMUS.

Synonym.—Lacmus.

Litmus is a blue pigment, obtained from various lichens, chiefly Roccella tinctoria, DC. (Cape Verde), R.

Montagnei, Bel. (Madagascar and Mozambique), and Dendrographa leucophaea, Darbish (California) (N.O. Discomycetes). The coarsely powdered lichen is mixed with pearl ash and solution of ammonium carbonate and submitted for several weeks to a slow process of fermentation, during which a red coloring matter is produced, which gradually changes to blue. Chalk and gypsum are then added, the mixture is passed through a sieve, then formed into small rectangular cakes and dried. Litmus occurs in dark blue or bluish-violet, finely granular, friable, and slightly aromatic, rectangular cakes. To prepare a sensitive indicator, commercial litmus is treated as described under Solutio Litmi.

Partially soluble in water or alcohol, forming solutions with a deep blue colour [basic], which are changed to red by acids.

Constituents. —Litmus contains several colouring matters, viz., erythrolitmin,azolitmin, erythrolein, and spaniolitmin, of which azolitmin and erythrolitmin appear to be the chief, but they are probably not homogenous substances. The colouring matter upon which the use of litmus as an indicator depends is a feebly acid, red body, the salts of which have an intense blue colour. The lichens from which litmus is prepared contain lecanoric acid (R. tinctoria), erythrin (R. Montagnei), and orcin. Lecanoric acid is diorsellinic acid, and is converted by alkalies into orsellinic acid. Erythrin is erythrite orsellinate, and is converted into erythrite and orsellinic acid. All these substances are colourless. Orsellinic acid yields by further change orcin, from which, by the action of air in the presence of ammonium carbonate, the colouring matters are produced. These appear to be oxidation products of amino-orcinol. Litmus also contains large quantities of chalk and gypsum.

Uses. —Litmus is much employed as an indicator; its colour is changed to red by acids, and the blue colour is restored by alkalies. As its colour is affected by carbonic acid, titrations in which carbon dioxide gas is liberated are better conducted with methyl orange as the indicator (helianthine). Methyl-orange solution is prepared by dissolving 0.2 of methyl orange in 2.5 of alcohol and sufficient distilled water to produce 100. It is comparatively unaffected by carbon dioxide, hydrogen sulphide, or hydrocyanic acid, but should riot be used for titrating organic acids.

2. mono , di , tri basi lactonic acid 3. Polyhydric [tetra to hepta ]alcohol 4. Triterpinoids (zeorin series) 5. Pulvic acid derivatives similar to Phenylbenzoquinone of higher plants 6. depsides like orcinol

7. quinone pigments like anthraquinones 8. Lichexanthone 9. dibenzofuran 10. Diketopiperazine

Uses: It has wide application

� Food and food adjunct: � Medicinal � Dyes

Litmus D H S Richardson: London, 1975. The lichens are ground in a solution of sodium carbonate and ammonia. Stir the lichens from time to time and the colour changes from red to purple and finally blue after about four weeks. The lichens are then dried and powdered. At this stage the lichens contain partly litmus and partly orcein pigments. The orcein is removed by extraction with alcohol, leaving the pure blue litmus.

� Parmelia spp. MISC. HUMAN USE India Crude drug 'chharila', extracted from 3 spp. of Parmelia is sold in Indian bazaars and used in Ayurvedic and Unani systems of medicine. Considered to be a good cephalic snuff.

� Parmelia spp. MISC. HUMAN USE India

� Crude drug 'chharila', extracted from 3 spp. of Parmelia is sold in Indian bazaars

and used in Ayurvedic and Unani systems of medicine. Considered to be aphrodisiac.

� Parmelia spp. MEDICINE, APPLIED EXTERNALLY India

Crude drug 'chharila', extracted from 3 spp. of Parmelia is sold in Indian bazaars and used in Ayurvedic and Unani systems of medicine. Powdered drug applied to

wounds.

� Parmelia spp. MEDICINE, TAKEN INTERNALLY India Crude drug 'chharila', extracted from 3 spp. of Parmelia is sold in Indian bazaars and used in Ayurvedic and Unani systems of medicine. Carminative and aphrodisiac; has been considered to be useful in dyspepsia [stomach disorders], spermatorrhoea[ degenration of sperm], amenorrhoea[ discharge of amines], calculi [formation of stone], diseases of the blood and heart, enlarged spleen, bronchitis, bleeding piles, scabies, leprosy, excessive salivation, soreness.

1. Reference: Saklani, A.; Upreti, D.K. 1992. Folk uses of some lichens in Sikkim. Journal of Ethnopharmacology 37: 229-233. Page 229.

Lecture 25

January 12, 2005

Leguminosae Acacia farnesiana (L.) Willd. Sp. Pl. iv. 1083. Reg. Trop

Shaini Perfumes oil, Hindu marriage,

Leguminosae Acacia arabica Willd. Sp. Pl. iv. 1085.

Africa,Asiatic tropic

Babool Babool gum, Gundpak, Sweets formulation

Fodder in xeric climate,tooth brush

Family: Leguminosae

Scientific Name: Acacia catechu Wild.

Sp. Pl. iv. 1079. Nepali: Khaira, Kattha, ; English : Cutch tree.

Leguminosae Acacia concinnaDC. Prod. ii. 464. Africa,Asiatic tropic

Shikakai Herbal shampoo

Plant distribution: Tropical to sub tropical dry xeric valleys at altitude of 100-2000m

Plant description: It is a moderate sized tree, 9-12 m high, with a dark colored bark and rough young shoots dark brown or purple, glabrous. Leaves bi-pinnate, 10-15 cm long main rachis pubescent, with glands between many of the pairs of pinnae.

Legal status: This is one of the oldest phyto-chemical industry in Nepal which is at the verge of closer due to shortage of raw material. The species is threatened (IUCN) and felling of the tree and exporting of raw logs is banned by HMG. The since 2057 BS.

However appears in forest revenue statistics [?].Royalty rate is NRs. 10/kg.

Trade status:

055/056 056/057 057/058 058/059 059/060 NA NA 12300 NA 11315

Bihar and UP are the main markets for Nepalese “kattha” .The price of “kattha” in a township in north Bihar was NRs 960/- to1120/-in 1991.

The price of “kattha” and cutch were NRs 1000/- and NRs 600/- per kg respectively in 1999.

Uses:

The bark and heartwood of the tree are used for many purposes.

Logs of Acacia catechu are used for religious purpose as firewood in “Havan”.

Most popular use includes: As “pari” along with slaked lime in Betel chewing, Gutaka & panparag.

Ayurvedic formulation: Kath-bol, Khadiradivati Khadirashtaka kashayam Khadirarishta

Pharmcologiaclly established property: Antioxidant property of aqueous extract of acacia catechu was compared by G. H. Naik, K. I. Priyadarsini, J. G. Satav, M. M. Banavalikar, D. P. Sohoni, M. K. Biyani and H. Mohan (2003). Antioxidant property was found lower than Terminalia chebula.

List of chemicals:

Major compounds Part of plant Concentration in ppm Carbohydrate Leaf 726000 Protein Leaf 130000 Fat Leaf 46000 Fiber Leaf 226000 Ash Leaf 98000 Calcium Leaf 27400 Phosphorous Leaf 1700 Catechins Exocarp 25000 to 330000 Epicatechins Phlobatanin 20-30 % Others: Gum Tannic, Gallic acids, Quercetin, Rutin, Flavones and its derivatives, and Sugars: like arabinose, galctose, Xylose Boron, Cobalt

Extraction: [Traditional process of extraction]:

catechu roots ----------chipped --------- cooked in water ---------filter -------------concentrate ---------allow to stand for weeks ----------supernatant water--- decant --- dry cakes of 0.5 cm thick dried in sun /oven ---- ready for market

Cutch is a bye-product of kattha production.

Epicatechin forms white acellular crystals, which are soluble in hot water and alcohol and give a green color with ferric salts.

Catechutannic acid is an amorphous phlobatannin, which is formed from epicatechin by loss of the elements of water. It readily yields the phlobaphene catechu - red.

Lecture 26

Scientific Name: Daphne bholua Buch. -Ham. Ex D.Don Prod. Fl. Nep. 68. Daphne bholua Buch.-Ham. ex D.Don subsp. emeiensis

Originally it was called Daphne cannabina

Family: Thymelaeaceae

Local name: Lokta, baruwa, seto baruwa etc

Plant Distribution: In the wild, Daphne bholua , grows in the Eastern Himalayas from East Nepal to Bhutan and Sikkim at altitudes of over 6000ft to 10,000ft and may reach up 18ft or so tall. At lower altitudes as an evergreen, it is normally found at the edge of thickets but the deciduous plants at higher altitudes may be found in grassy down-land-like areas.

Plant Description:

Deciduous, evergreen or semi-evergreen shrubs; Flowers almost white to dark red-purple; scent, flower size and plant habit variable

Not much is clear among trade circle but while working with Dolkha collectors of Lokta bark we have noted that there is some notable difference among fiber and its quality dependent on altitudes and location of collection 1998.

Plant with flowers

Bast fibers

This is now clearly stated in following paper of Royal Horticultural Society (2004)

Name Description History

‘Alba’ Evergreen to semi-evergreen; bushy with narrow leaves; sometimes undulate; flowers with pale pink flushed buds which open white or with more or less white buds.

Possibly should be f. alba but there is no publication for this name. Chris Brickell hopes to publish it in an article in New Plantsman this year. ‘Alba’ is an invalid name but there are at least 2 forms in cultivation as ‘Alba’. One has pale pink buds and outside of petals opening white. One plant from Wisley received from Burncoose has the least pink flush on the flowers and buds. The plant from Robin White was the best exhibited but unfortunately most of others were rather small cut pieces. Some of the palest flowered Peter Smithers seedlings were not dissimilar. Best and distinct forms should be given cultivar names. Perhaps the best should be described and given a cultivar name.

Daman Ridge/ Peter Smithers seedlings

Deciduous, evergreen or semi-evergreen shrubs. Flowers almost white to dark red-purple; scent, flower size and plant habit variable.

Sir Peter Smithers 1971 or 1972 collected a number of plants from Daman Ridge, Nepal at 9000ft. Seed has been collected for distribution from these plants growing in Switzerland and sent to a many gardens and individuals. A large number at Wakehurst show the complete range of characteristics and one or two have been selected and named, such as ‘Peter Smithers’. Not a distinct cultivar but a range of seedlings and seedlings of seedlings from original plants collected on Daman Ridge. Best called simply Daphne bholua followed, if necessary, by some sort of descriptive explanation that these are seedlings which originated from plants collected by Sir Peter Smithers from Daman Ridge, Nepal. A group name would be inappropriate as there is no way of defining such a morphologically wide range of plants and it would be unwise to call them "Peter Smithers seedlings" because of the possible confusion with the cultivar ‘Peter Smithers’. "Daman Ridge seedlings" would indicate that they were first generation seedlings which of course many are not.

‘Daman Ridge Dark’

Very dark red-purple buds see ‘Peter Smithers’.

Selected in 1990 at Wakehurst by Robin White for darker flowers from Peter Smithers seed sent to Wakehurst . Considered to be same as ‘Peter Smithers’ and R White suggested he market his plant as ‘Peter Smithers’ and drop the name ‘Daman Ridge Dark’ which is not yet being sold.

‘Darjeeling’ Bushy evergreen or semi-evergreen shrub. Stems with slightly downy appearance. Pale pink to white flowers, very early flowering, often November to December but not invariably. Slightly less hardy than others.

Collected from a plant on south wall of Curators house at Wisley at least 15 years ago; propagated by Hinton Nurseries; distributed initially from Wisley Plant Centre but later by other nurseries such as Starborough. Original plant was received from Ghose of Darjeeling, hence cultivar name, in 1961 and was 3m tall in 1976. (conversation with Chris Brickell 1-00). The original plant is no longer alive at Wisley. One of easiest to grow from cuttings. Recent discussions with Brian Humphrey have highlighted the fact that the flowers of young plants may be slightly darker in colour and may not open white. The corolla tubes of the flowers of young plants are slightly more purplish pink whereas those of the more mature plants are a more pale rose pink colour.

‘Glendoick’ Flowers larger and with a richer colour than ‘Jacqueline Postill’ and plant appears to be tougher.

Not exhibited but will be listed in the next edition of The Plant Finder. Propagated by Brian Humphrey from material received from Peter Cox. Name was originally to be ‘Heaven Scent’ but this name has not been released and not published and changed at request of raiser.

‘Gurkha’ Deciduous, tall, fairly upright shrub. Very fragrant, dusky mauve pink and white flowers.

Major Tom le M Spring Smyth 18 March 1962 on the Milke Dhanra ridge, E Nepal, 10,000-10,500ft. Growing in open grassy terrain with Rhododendron arboreum and R. barbartum. Collector’s number TSS 132B. Named ‘Gurkha’ as the collector was an Gurkha officer. Several specimens exhibited were incorrect and ‘Gurkha’ should always be deciduous. Illustration and description of collecting in The Garden (1976) article by Roy Lancaster, p. 454-457. (AGM 1993).

‘Jacqueline Postill’

Evergreen or semi-evergreen, vigorous, upright shrub. Large bright rosy pink, very fragrant flowers.

Selected in 1982 from 3 (self pollinated) seedlings of ‘Gurkha’, the only one which was not deciduous, by Alan Postill at Hilliers Nursery and named after his wife. (FCC 1991, AGM 1993). Growing conditions appear to affect the stem colour as plants growing vigorously appear to have a more grey bark than the brown bark seen in other plants propagated from the same parent stock.

‘Peter Smithers’

Evergreen or semi-evergreen shrub. Buds dark red-purple opening darker than other evergreen

Selected as a very dark flowered plant by Chris Lane and propagated by Colin Tomlin of Starborough Nursery about 1993 from the generations of Sir Peter Smithers seedlings from original plants collected from Daman Ridge, Nepal at Wakehurst. The original dark one has died at Wakehurst according to a letter from Tony

cultivars. Schilling (2-2-00). Considered by committee to be the same as ‘Daman Ridge Dark’ and this name retained. The plant was first listed in Starborough catalogue in about 1996. (AM 25 January 2000).

‘Rupina La’ Deciduous with rather thick stems. Very large, long dark purplish flowers. Best grown as a conservatory plant except in mildest areas.

Collected by Tony Schilling 3-April-1983,(Schilling 2611) N.E. Barbak, Gurkha Himalayas, Central Nepal at c. 9000ft. Selected from 9 seedlings and raised at Wakehurst. Deciduous but not as hardy as other named cultivars. Illustrated in the wild in The Garden (1985) article by Tony Schilling, p170-174.

‘Sheopuri’ Semi evergreen to evergreen, compact shrub. White flowers slightly flushed purple at base; buds pale pink.

Selected from seeds collected by Dr G Herklots 1962. The original plant was at Windsor (AM 1973) but now dead and there are probably no others in cultivation. It would probably fit into f. alba but none of the existing plants of "Alba" have the same combination of small flowers and broader leaves.

Wakehurst A selection from ‘Sir Peter Smithers seed but no description of an exact cultivar and the name appears to be used for different seedlings. Not a named cultivar but a name given inappropriately to plants from Wakehurst.

acutiloba x Bholua (male)

Evergreen bushy shrub, probably hardier than bholua with smaller but spicy scented flowers. Leaf undulate with prominent midrib on under surface.

Raised by Robin White and to be described as a new hybrid by C D Brickell in issue of The New Plantsman 2000.

‘Gurkha’ x ‘Alba’ (male)

Vigorous semi evergreen to evergreen shrub. Like ‘Jacqueline Postill’ but paler in colour

Raised Mark Fillan

Related species 1a Fruits black; some purple color on flowers bholua

b Fruits red fruits and white or greenish white flowers

2

2a Flowers not fragrant and leaves dull dark green

papyracea

b Flowers fragrant; leaves pale shiny green (not very hardy)

sureil

Part used: Some cultivars, which bloom in winter and have scented flowers are ornamental garden plant in England and Switzerland. [Case of genetic piracy through research]. Flower, fruit, seed and roots are in traditional use but principal part used is inner bark or bast fibers.

Collection of Lokta for bast fibers:

Main collection districts are:

Eastern: Panchthar, Taplejung, Bhojpur, Sankhuwasabha, Khotang Okhaldhunga, Solokhumbu,

Central: Lalitpur,Dhading,Sindhupalchok,Ramechap,Dolkha

Mid western: Parbat,Banglung,Myagdi,

Western: Jajarkot, Salyan, Rukum,

Far western: Bajhang ,Baitadi

056/057 057/058 058/059 059/060 208518 233912 NA 144614

Market price in the mountain village in 1999 was NRs. 24/kg. Kathmandu price was 65/-

Medicinal uses: Medicinal uses of bark, seeds, root for fever, and anthelmintic and intestinal troubles.

According to Manandhar, N.P. (1995) and Bhattarai, N.K. (1989) bast fibers made papers are boiled in water a gelatinous mass, and are applied to treat micro-fracture or dislocated bone.

In terai the hand made papers are applied on boils [wounds] along with honey.

Some other uses in villages are: Rope making.

Traditional Nepal Paper:

Textured hand-made, deckle edged papers produced from bast fibers. Chemistry of Bast fibers: These are not secondary metabolites but are composed of Lignan and cellulose [82%] Singh (1978).

Production of Nepali paper:

Bark is taken out from plants with basal diameter 2 to 3”. These barks are peeled for inner bark called Bast fibers. These bast fibers are digested with quercus [banjh] ash or caustic soda. It is then beaten with wooden hammers to obtain the pulp and washed with sufficient water to make it alkali free. The alkali free pulp is spread in water tank and spread to thin layer and then filtered with framed sieve. This still moist layer is dried in sun to obtain Hand made Nepali paper.

The paper sheet is marked at rate of 200/- to 1200/- per 200.

Trade from Nepal:

� Nepal export of handmade uncoated paper [HS code 480210] was 1% of total global trade 941 ton worth US$ 1000. Nepal’s ranking in the global trade is 13th in the year 2003.

� Major importers are India [46%], US [17%], France [15%], Japan [6%], Germany [5%], Denmark [2%], Switzerland [2%], Greece [2%], Italy [1%], Netherlands [1%], Canada [1%].

� The price per ton was found to maximum in Germany [26000 $] , Denmark[23000$] and Switzerland [21000$] and minimum in India [521$] .

Lecture 27

"Shilajit"

Hindu texts. "Shilajit" (properly pronounced Sil'-i-jeet") literally means "conqueror of mountains & destroyer of weakness" in Sanskrit.

Charak, described “Shilajit” as a substance which assists the cure of all shorts of disease.

A bituminous substance found in the lower Himalayan Mountains.

In its native state, Shilajit is a compact mass of vegetable organic matter composed of a dark gummy matrix - thought to be largely the result of humification of the plant Styrax officinalis Linn and/or Euphorbia royleana Boiss and is gathered from stone formations, either on the ground or from fissures in the rocks. Before refinement, Shilajit is a semi-hard, brownish black to dark, greasy, black resin that has a distinctive smell and taste.

Shilajit is primarily composed of fulvic acid. Fulvic acid is the end product of nature's humification process. It contains all of the phytochemical protective substances, amino acid peptides, nucleic acids, etc. from the original plant matter.

Euphorbiaceae Euphorbia royleana Boiss. in DC. Prod. xv. II. 83.

Euphorbia royleana Boiss.

Formation of Shilajit : The latex ,the milky exudents of the plant Euphorbia royleana when absorbed in adjacent rocks and mineral ‘Shilajit” is formed . And accordingly can be classed according to the mineral present.

� Golden shilajeet is yellowish and is consid- ered to be of the best quality. � Silver shilajeet is whitish and next best type. � Copper shilajit are brownish � Iron shilajits black or iron shilajeet. The last one is considered of poorest quality. Distribution: Mugu, Dolpa, Jumla, Kalikot , Myagdi ,Darchula are the districts from which hit is being collected. Legal status: The trade of shilajit in unprocessed form is banned by HMG Nepal gazette 2058. However it appears in tread: 055/056 056/057 057/058 058/059 059/060 10853 47365 13042 4791 Unit price per kg in the year 2040 55/- in Dang and current price is NRs 185 /- to 200/- per kg. Price of pure processed shilajit in Indian market is found to be INR 1100/- +

Chemistry: Fulvic acid is the major constituent shilajit besides over dozen aminocids are known. Fulvic acid is a complex molecule, which can’t be synthetically imitated.

It is poly hydroxy molecule even the scientific naming of this molecule has not been tried.

Some people attribute pharmacological property to co-ordination compound formed minerat atom like Gold, Silver, Copper, or Iron.

We do not want you to try the structure

The complex structure of components gives opportunity of marketing of shilajit from natural resources.

Pharmacological

Bani S, Kaul A et al (2000) has found dose-dependent anti-inflammatory and anti-arthritic effects in hydro-soluble fraction of Euphorbia royleana latex on different acute and chronic

test models in rats and mice.

Mahadev Rao (1997,1999) has noted anti-tumor activity in “polyherbal formulation cystone” in cisplatin induced nephrotoxicity of mice and renal problems of rats.

R. Sundaram (1996), investigated D-400, a herbomineral preparation for its interaction with oral hypoglycaemic agents namely, tolbutamide and glibenclamide in alloxan-induced diabetic rabbits and has proven antidiabetic activity in experimental models as well as in clinical trials. Abdel-Hamid HF (2003) Molluscicidal and in-vitro schistosomicidal activities of the latex and some extracts of some plants belonging to Euphorbia. the lyophilized latex extract of E. royleana gave 100% mortality after exposure to 30 ppm for 4 consecutive days and 66.7% mortality after exposure to 10 ppm for 5 consecutive days. Mitra SK, (1996) has investigated the effect of D-400, a herbomineral formulation on liver glycogen content and microscopic structure of pancreas and liver in streptozotocin induced diabetes in rats. Vaishwanar I, (1976) has studied changes in liver and serum lipids of Shilajit in carbon tetrachloride induced liver damage.

Lecture 28

Terminalia sps

Family: Combretaceae

Terminalia chebula Retz.

Terminalia chebula Willd. ex Flem.

in As. Res. xi. (1810) 181.

Harro

Terminalia bellerica Roxb. Barro

Terminalia arjuna Wight & Arn. Prod. 314, in textu; Bedd. Fl. Sylv. I. 28.

Arjuna

Scientific Name: Terminalia chebula Retz. Local Name: Harro, Jangali harro, Thulo harro English Name: Also known as yellow or black myrobalan or Chebulic myrobalan Distribution: Tropical and sub-tropical forests between 200 –700 m.

Description: Medium sized deciduous tree. Best time of collection is December – February. Parts used: Mature and immature fruits Collection and trade: Mostly Terminalia sps are collected from terai districts Of the country principally because they grow there and market price is low which makes it economically unviable from hills. 16,991 kg has been collected in the year 059/060 Market Information: Price at market centers is NRs. 16/kg (1997). Royalty for dry fruits is NRs. 2/kg. Properties and Uses: The fruits are astringent, sweet, acrid, bitter, sour etc. Bark, fruits and seeds have medicinal uses such as for skin diseases, leprosy, and intermittent fever, cardiac disorders. Chemistry: Juang LJ, Sheu SJ, Lin TC. (2004) Isolated fourteen components (gallic acid, chebulic acid, 1,6-di-O-galloyl-D-glucose, punicalagin, 3,4,6-tri-O-galloyl-D-glucose, casuarinin, chebulanin, corilagin, neochebulinic acid, terchebulin, ellagic acid, chebulagic acid, chebulinic acid, and 1,2,3,4,6-penta-O-galloyl-D-glucose) in the fruit of Terminalia chebula Retz..

Pharmacology:

The antioxidant activity: Cheng HY, Lin TC, Yu KH, Yang CM, Lin CC. (2003) Free radicals react with biological molecules and destroy the structure of cells, which eventually causes free-radical induced disease such as cancer, renal failure, aging, etc. In this study, 6 extracts and 4 pure compounds of Terminalia chebula RETZ. were investigated for anti-lipid peroxidation, anti-superoxide radical formation and free radical scavenging activities. The superoxide radical scavenging of the 4 pure compounds was further evaluated using electron spin resonance (ESR) spectrometry. The results showed that all tested extracts and pure compounds of T. chebula exhibited antioxidant activity at different magnitudes of potency. The antioxidant activity of each pure compound was derived from different pathways and was suggested to be specific.

Antioxidant: Naik GH, Priyadarsini KI, (2004) The aqueous extract of T. chebula acts as a potent antioxidant as it is able to protect cellular organelles from the radiation-induced damage.The compound such as ascorbate, gallic acid and ellagic acid present are responsible for the activity. Activity on oxidative stress: Na M, Bae K, Kang SS, Min BS, Yoo JK, Kamiryo Y, Senoo Y, Yokoo S, Miwa N. (2004) noticed inhibitory activity on oxidative stress and the age-dependent shortening of the telomeric DNA length in the the ethanol extract of fruit of Terminalia chebula.

Cardioprotective effect: Suchalatha S, Shyamala Devi CS. (2004) proved the protective effect of ethanolic extract of Terminalia chebula on cardiac muscles.

Inhibition of cancer cell growth: Ammar Saleem et al (2002) has shown the inhibition of cancer cell growth by the phenolics present in fruits of Terminalia chebula . , compound responsible are gallic acid, ethyl gallate, luteolin, and tannic acid. Chebulinic acid.

HIV-1 : Ahn MJ, Kim CY, Lee JS, Kim TG, Kim SH, Lee CK, Lee BB, Shin CG, Huh H, Kim

(2002) galloyl glucoses from Terminalia chebula Inhibits of HIV-1 integrase

===========

Lecture 29

January 16, 2005

“AMLA” Scientific Name: Emblica officinalis Gaertn. Fruct. ii. 122. t. 108. f. 2.,

Original name Phyllanthus emblica L. Sp. Pl. 982.

Family : Euphorbiaceae

Nepali Name: Amala ; English Name: Indian gooseberry Distribution: Normal distribution Asiatic tropic ; In Nepal it is available up to 1400 m in Churia and mid-hills. Description: Large deciduous shrub. Fruit can be collected April – August. Market Information: Royalty of the dry fruit is NRs.2/kg. Dried fruit is traded at road heads for NRs. 20/kg (1999) and NRs. 34/kg (2001). The price fluctuation is often seen in all tropical crops like Amla because the productivity is often low in alternate years hence net global supply is low in alternate years but the demand is in + trend.

Collection and trade: Sindndhuli,Rauthat,Parsa,Nuwakot,kathamndu,Chitwan are the major districts from where forest statistics appears. 055/056 056/057 057/058 058/059 059/060 38385 4394 100 NA NOT

Recorded

The statistics shows neither significant quantity, nor regularly Amla is being collected, though it is one of the recipe of “TRIFALA” and “Chyawanpras” and well known to many people through out the population.. Ayurvedic companies producing “ Chyanwanpras” are major consumer of Amla. But the Nepal Amla has been found incompetent in price to those produced from Horticultural improved crops of central India. Mostly native industries and vaidyas are the actual consumer of local wild Amla. Parts used: Fruits Uses: Fruits are sour, edible and rich source of vitamin- C. Besides it is commonly used as pickle, candied [Morabba] form.

Medicinal uses such as cooling, refrigerant, diuretic and laxative. Raw fruit is aperient. Chemistry: Preliminary phytochemical screening of the extracts showed the presence of alkaloids, tannins, phenolic compounds, carbohydrates and amino acids.

Pharmacology:

anti-pyretic and analgesic activity: Perianayagam JB, Sharma SK, Joseph A, Christina AJ (2004) attributes its anti-pyretic and analgesic activity alkaloids, tannins, phenolic compounds, carbohydrates and amino acids present in it.

Antioxidant property: L Anila and N R Vijayalaxmi attributes it antioxidant property to the flavonoids present.

Myocardial adaptation: Rajak S, Banerjee SK, Sood S, Dinda AK, Gupta YK, Gupta SK, Maulik SK.(2004) opines that Emblica officinalis causes myocardial adaptation and protects against oxidative stress in ischemic-reperfusion injury in rats.

Prevention of sugar cataract: Suryanarayana P, Kumar PA, Saraswat M, Petrash JM, Reddy GB. (2004) Inhibition of aldose reductase by tannoid principles of Emblica officinalis: implications for the prevention of sugar cataract.

Hyperthyroidism: Panda S, Kar A. noted that the ethanolic extract from the fruits of Emblica officinalis Gaertn ameliorates hyperthyroidism and hepatic lipid peroxidation in mice.

Cytoprotective activity: Sai Ram M, Neetu D, Deepti P, Vandana M, Ilavazhagan G, Kumar D, Selvamurthy W. (2003) Cytoprotective activity of Amla (Emblica officinalis) against chromium (VI) induced oxidative injury in murine macrophages.

anti ulcerogenic property: Same author (2002) has recorded that Emblica officinalis extract had significant ulcer protective and healing effects and this might be due to its effects both on offensive and defensive mucosal factors.

ischemia-reperfusion-induced oxidative stress in rat heart: Bhattacharya SK, Bhattacharya A, Sairam K, Ghosal S. shown the effect of bioactive tannoid principles of Emblica officinalis on ischemia-reperfusion-induced oxidative stress in rat heart.

Hepatoprotective activity of Emblica officinalis Jose JK, Kuttan R.and Chyavanaprash (2000) found Hepatoprotective activity in Emblica officinalis extract.

Section IV Isolation, purification and identification of active ingredients: Pulverization, extraction Distillation, Solvent extraction, Solvent-solvent extraction, Crystallization and re-crystallization, T.L.C., Column chromatography; G.C., HPLC; Analysis of active ingredient of above selected Species: Application of modern analytical methods. M.P.; B.P.; refractive index, Optical rotation, Flash point, Co-TLC; G.C.; U.V.; I.R.; N.M.R.;

LatinName PlantPart Grade Therapeutic Uses

1. Acacia catechu Wood Tannins 60% Powerful astringent, Chronic diarrhoea, Leucorrhoea 20-60%

2. Acorus calamus Rhizome Sedative, Tranquilizer, Antihepatotoxic, Antispasmodic

3. Adhatoda vasica Leaf Alkaloids 2% Expectorant, Antispasmodic

4. Aegle marmelos Fruit Chronic dysentery, Astringent, stomachic

5. Albizzia lebbeck Bark Coumarin Abortifacient, Restorative, Protects against histamine bronchospasm

6. Allium cepa Bulb Quercetin 4% Cardiotonic, antidiabetic, hypolipidemic, Aphrodisiac, antithrombotic

7. Allium sativum Bulb Allicin Min 2.5% Antitumor, anticholesterolemic, Antihypertensives, Rejuvenator

8. Aloe indica Gel Aloin 1% Emmolient, Improves menstruation

9. Andrographis paniculata Entire plant 8% Andrographolides

Antipyretic, Alterative Hepatoprotective

10. Asparagus racemosus Root 20% Saponins Aphrodisiac, cooling, Antioxytocic, Hypotensives

11. Asphaltum Others WSE 70% Aphrodisiac, diuretic

12. Melia azadirachta Leaf Bitters 3% Antiviral, antileprotic, Diuretic, Insecticide

13. Melia azadirachta Seed Per % Aza Content Pesticidal, antifeedant

14. Berberis aristata Alkaloids 8% Root Antiamoebic, Deobstruent, Antihypertensive

15. Bergenia ligulata Root For bladder stones

16. Bixa orellana* % Bixin Seed Natural Color

17. Boerhavia diffusa Root Total alkaloids 0.08%

Diuretic Anti inflammatory

18. Camellia sinensis

Leaf Poly-phenols 60% Astringent, Stimulant

19. Carum carvi Fruit Mucilage, essential oil

Carminative, Mild stomachic

20. Cassia anguistifolia Leaf and pods Per % Sennosides Laxative

21. Cassia fistula Fruit Laxative, Anti-fungal, astringent

22. Citrus aurantifolia rind Fruit Flavones 5-6% Source of vit 'P', Anti acne Bitter tonic

23. Convolvulus pluricaulis Entire plant Bitters 1.5% Brain tonic, Antihypertensive, Tranquiliser

24. Costus speciosus Root/Rhizome Alkaloids 0.4% Purgative, Tonic, Astringent

25. Cucurbita pepo Seed root Amino Acids 4% Anthelmintic, Nervine stimulant

So now, we know that secondary metabolites are distributed in Root, Rhizome, Bulb, Stem, Leaf, Wood, Bark, Flowers, Fruits, Seeds or almost any where in plant. Further, these compounds can be terpines, alkaloids, steroids, glycosides etc and remain present in deep into the heart wood of a tree species or surface of leaf or fruit pericarp. Hence their process of extraction is dependent on where they are present or what type of compound they are. If from our traditional knowledge it is known which part are in use then it can be used as information for processing if not then we look for Phyto-chemical screening. Lecture 30 Date January 19, 2005 Secondary metabolites may be lodged in Root, Rhizome, Bulb, Stem, Leaf, Wood, Bark, Flowers, Fruits, Seeds or almost any where in plant. Further, these compounds can be terpines, alkaloids, steroids, glycosides etc and remain present in deep into the heart wood of a tree species or surface of leaf or fruit pericarp. Hence their process of extraction is dependent on where they are present or what type of compound they are.

If from our traditional knowledge it is known which part are in use then it can be used as information for processing if not then we look for Phyto-chemical screening. Cutting into small pieces /slices then dried in oven preferably below 50 0 c or in shade. Direct sun is avoided: Why direct sun is avoided? Direct sun is avoided because in plants we may have any molecular structure say alkaloids:

They are all nitrogen heterocycles which occur mainly in plants as their salts of common carboxylic acids such as citric, lactic, oxalic, acetic, malic and tartaric acids as well as fumaric, benzoic, aconitic and veratric acids. These alkaoids have teriary nitrogen, which can intercat with atmospheric oxygen to give oxygenated compounds. They are not dried at higher temperature above 500C because some bioactive compound decompose by interaction with water molecule present there in or interact with O2 present. Once the crude plant material is dry enough [with moisture content less than 8% to 12%] to be grinded, they are grinded. Grinding / pulverize/ crush/chop is common process both at home and or laboratory or industry where Grinding stone “silouto”, “Janto” like mortar pestle, or mixie, or grinding mill of any capacity as desired up to few ton per hour grinding capacity. The purpose of grinding etc is to increase the surface area of the material to exposed to appropriate solvent. Sieving /strainer /shifter devices that are common at house as “Chalnu”,”chiya channe”. In laboratory / industry it is desired so that the packed material can be as

2 to 5 M

High as 5 meters and if the powder is very small and compact they may hinder the flow of solvent through it. Extraction / taking out is also a common process at household activity: Squeezing of juice from lemon manually or by mechanical juicer or elector-mechanical juicer. Taking out of oil: by Chepuwa / expeller /scew press / hydrolic press / Then we can think of using solvent: solvent can be cold, hot, boiling, vapors of solvent. Solubility we know is dependent on temperature:

Temperature vs solubilty

0102030

405060

1 2 3 4 5 6

solubility

tem

pera

ture

Temperature

Tea making at home or extraction by solvent [H2O] boiling; Instead we can use any organic solvent that takes out the particular chemical “Caffeine in case of tea”. Solvent: Dielectric constant is an important physical property used in the process of extraction you can see how pentane with dielectric constant 1.8 and water with Highest dielectric constant 80.2

Dielectric constant

020406080

100

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Ca

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Eth

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Name of solvent

die

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Low polarity ---------------------- High polarity So when we are in need of solvent with low dielectric constant we choose from left and when we need solvents of high polarity we choose from right side. The solvent series above is used in

1. Solvent extraction 2. Solvent-solvent extraction 3. Crystallization and re-crystallization 4. Thin layer Chromatography 5. Column chromatography and 6. High pressure liquid chromatography

Cold extraction: when the entire process of taking out with help of a solvent is carried out at room temperature 250C.

Hot extraction: Simple boiling with solvent or washing repeatedly with boiling solvent: For simple boiling we have tea-making at home.

Industrial extraction: Distillation:

Lecture 31 Date January 21, 2005 Extraction: continued: Percolators

Percolators lab Percolators industry

Hot extraction: [Soxhlet’s extraction] Soxhlet’s extraction in lab

Soxhlet’s extraction industry image

Solvent recovery & Recycling: Extract obtained contains large amount of solvent which are many time not desirable hence both in lab and in industry solvent need to be taken out. This is concentration or recovery of solvent. When we are repeating the process of extraction for the same plant and solvent is single not a combination of two or more we can re-use the solvent. The case is not same for research where trace of contamination from previous lot may mislead the result.

Now we have the extract it may have n number of constituents: Example:

Rauvolfia serpentina (L.) Nepali name “Sarpagandha” “Chandmaruwa “ root has total alkaloids content between 15000 –24000 ppm. If we fractionate it these compounds are over 37 in number. Even those majors are ajamlexine 2000 to 3000ppm,Resajmaline 5000 to 8000ppm Rescinnamine 25 to 75 ppm,Reserpine 100 to 2000ppm and serpajmaline 13000 to 16000 ppm

� Extract from low polar to high polar � Ethanolic extract then fractionate in to extract of different polarity [lower – to higher]

Vacuum concentration:

Vacuum concentration in lab

Vacuum concentration in Industry

Vacuum and its effect on boiling These processes use a number of solvents like benzene, chlorinated hydrocarbons like dicholoromethane, chloroform that now have been found unfriendly to environment.

Clean tech & clean solvents: There is a great deal of interest in replacing traditional solvents with 'clean solvents' in industrial processes. Two solvents currently in the frame are carbon dioxide and water. Already certain large-scale processes are benefiting from their use, including the decaffeination of coffee and extracting hop oils for beer production.

Carbon dioxide and water often have to be put under special conditions to make them useful solvents. Carbon dioxide, a gas under ambient conditions, must be used under pressure to convert it to a liquid or supercritical solvent. The carbon dioxide used for this purpose is a byproduct of other processes, such as fermentation and ammonia production, and does not contribute to the greenhouse effect. Water, which is highly polar at room temperature, must be heated under pressure to temperatures at which it becomes less polar, to dissolve many organic compounds. Both solvents have the advantages of being inexpensive and pose little hazard in use.

Let us consider the physical basis for using these solvents, and some applications.

Liquid carbon dioxide

Fig 1. Phase diagram of CO2

1 Pascal = a force of 1 Newton per meter2 (1 Newton = the force required to accelerate 1kg one meter per second per second = 1 kg.m/s2 ; 1 MPa = 1,000,000 Pascal

Figure 1 is a phase diagram of carbon dioxide The pressure axis is nonlinear and the solid phase at the higher temperatures shown occurs at pressures of the order of GPa. The areas in which the substance exists as a single solid, liquid or gas phase are labeled, as is the triple point where the three phases coexist. If we move along the gas-liquid curve, both temperature and pressure increase. The liquid becomes less dense because of thermal expansion and the gas becomes denser as the pressure rises. Eventually, the densities of the two phases are identical, and the distinction between the gas and the liquid disappears at the critical point. The substance is now described as a fluid. (The fluid is like a gas at lower pressure and like a liquid at higher pressures.) The critical temperature and pressure for carbon dioxide are 31°C and 7.4MPa respectively. Below 31°C carbon dioxide can be used as a liquid, provided the pressure in the system is sufficient, which is ca 5MPa at ambient temperatures. Above 31 °C carbon dioxide is a supercritical fluid at pressures above 7.4MPa.

That there is a critical temperature, above which a single substance can exist only as a fluid, and not as either a liquid or gas, can be shown experimentally. The experiment is based on one originally described by Baron Charles Cagniard de la Tour.1 A metal cell with thick glass walls,

Fig 2. Experimental set-up to show the critical temperature of a liquid fluorocarbon

Fig 2, contains a liquid fluorocarbon of critical temperature 95°C. The cell has an outer jacket, through which steam is passed to raise the fluorocarbon to its critical temperature. Shown below are three photos: the left picture is taken at the lowest temperature and shows the liquid and gas phases with a meniscus between them; as the temperature and pressure rise, the density difference between the two phases becomes less and the meniscus becomes less distinct (shown in the middle picture) though in practice the meniscus is no longer flat, because of temperature fluctuations and the small density difference; when the critical point is passed the meniscus disappears, as shown in the bottom right-hand photo.

Applications As well as being environmentally friendly - clean solvents reduce pollution and do not leave undesirable solvent residues - supercritical carbon dioxide has the added advantage of being a tuneable solvent. Above the critical temperature its density, and therefore other properties, can be varied continuously from gas-like to liquid-like. Solvating properties, for example, can be controlled, and thus extraction can be made selective to some extent. Other applications of this solvent include fractionation, chromatography, cleaning, dyeing, impregnation (eg of

photochromic compounds into plastic sunglasses), particle formation, metal extraction and chemical reactions. Some of these are in the research stage, while others are established industrial processes.

The major applications of carbon dioxide as a clean solvent are in the decaffeination of green coffee beans and the extraction of hop oils for beer production, principally for bittering beer. The removal of caffeine from coffee was originally done with chlorinated solvents, which were replaced by the more acceptable organic solvent ethyl ethanoate. The use of carbon dioxide in this process was developed in Germany in the 1980s, with the largest production plant in Houston, Texas, which measures 51m high. The coffee beans pass down a tower with carbon dioxide at ca 20MPa passing upwards. The process is semi-continuous with coffee beans entering the top in a compression chamber and passing out through a decompression chamber at the bottom.

Hop oils were extracted initially with liquid carbon dioxide using a process developed in the Brewing Research Institute in Surrey. Supercritical carbon dioxide is now more widely used in the hop industry because it extracts the resins and bitter compounds more rapidly.

Another application, though still at the initial research stage, exploits the tuning property of supercritical carbon dioxide, ie in controlling the product of a reaction. For example, in a reaction in which two isomeric products can be produced, changing the pressure and therefore the density of carbon dioxide solvent can change the proportion of isomers produced. The transition states leading to the products are stabilised by solvation, which is to an extent dependent on the density of the solvent. Thus at a particular density, the transition state leading to one product may be preferentially stabilised, leading to more of the corresponding product. Several reactions show this, but the most spectacular example is the pressure-dependent enhancement of diastereoselectivity for the sulphoxidation of a cysteine derivative,2 illustrated in

Fig 3. Pressure-dependent enhancement of diastereoselectivity for sulphoxidation of a cysteine derivative 2

Fig 3. As can be seen, by adjusting the pressure, an almost pure diasterioisomer can be produced. These initial results are scattered, owing to the difficulty in controlling conditions, but further work is being carried out on this important effect.

Superheated water

The critical temperature of water is 374°C. Water below its critical temperature but above 100°C is called superheated water. In recent years, superheated water has been considered as a replacement for organic solvents for extractions and other processes. Much of this work has been restricted to the 100-250°C range. At these lower temperatures, water is not highly compressible and the pressure of the medium has little effect, as long as it is high enough to maintain the water in the liquid phase. The pressures required for superheated water are typically not as large as for supercritical carbon dioxide. At 200°C the vapor pressure of water is 1.2MPa. Moreover, only 505kJkg-1 is required to heat liquid water from 30°C to 150°C, compared with 2550kJkg-1 required to convert water at 30°C to steam at 100°C, representing considerable energy savings.3

The solubility of an organic compound in superheated water is often many orders of magnitude higher than its solubility in water at ambient temperature for two reasons. First, water changes dramatically when its temperature rises because of the breakdown in its hydrogen-bonded structure with temperature. The high degree of association in the liquid means its relative permittivity (or its dielectric constant) is very high at ca 80 under ambient conditions, but as the temperature rises this falls, as shown

Fig 4. The dielectric constant (relative permittivity) of water as a function of T at its vapour pressure 3

Fig 4.3 By 205°C the dielectric constant of water is equal to that for methanol (ie 33) at ambient temperature. Thus, between 100°C and 205°C, superheated water behaves like a water-methanol mixture.

Secondly, a compound with low solubility at ambient temperature will have a high positive enthalpy of solution, and thus there will be a large increase in solubility with temperature. Compounds that show high solubility in superheated water are often polarisable, such as

aromatic compounds, or have some polar character. This allows a range of applications to be considered.

Applications of superheated water

The use of superheated water as a replacement for organic solvents is still in its infancy. However, some processes are close to exploitation. The higher solubilities of aromatics have made water a candidate for extracting solids and liquids from soil and environmental decontamination in general,4 and in chromatography. Extraction of environmental pollutants for monitoring and decontamination is being extensively studied. For example, superheated water is used to extract polyaromatic hydrocarbons and polychlorinated biphenyls from soil and sediment, and virtually all the material is removed at ca 250°C.5,6 This process has been done on a pilot scale with an eight-litre pressure cell.7 Usefully, after the extraction, much of the humic organic matter was still present in the decontaminated soil. For some samples, plants such as carrots thrived in the decontaminated soil, whereas they could not survive in the original soil. Decontamination of soil with superheated water can also be done in situ with a device that can be hammered into the soil on a contaminated site.8 The bottom of the device is open and the pressure vessel is formed either when the bottom seals into a clay subsoil, or by freezing the bottom layer with liquid carbon dioxide.

Superheated water is also being used to extract essential oils from natural products in the food and fragrance industries. For example, extracts of rosemary (Rosmarinus officinalis) using water at 100°C and 200°C6 as the solvent, contained oxygenated compounds, such as 1,8-cineol, camphor, borneol and linalyl propanoate. The extract at 200°C also contained small quantities of the monoterpene hydrocarbons α-pinene and camphene, but the extract at 100°C did not. The oxygenated compounds give flavor and fragrance to the essential oil, whereas the monoterpenes are the less valuable components. Thus extraction with superheated water below 200°C produces a more valuable extract of higher quality.9 These experiments could form the basis of a method for removing the terpenes from essential oils. This is currently done for citrus oils by cold pressing, a process that uses hexane as the solvent.

Superheated water can also be used to extract liquids. Because of the higher solubility of aromatics compared with saturated compounds, aromatic compounds can be extracted from petroleum products,10 which could provide a way of removing benzene from gasoline.

During any extraction, some of the components may undergo reactions, which may or may not be advantageous. In the extraction of explosives, for example, from decontaminated soil the explosives are degraded to benign substances.11 For plant materials at lower temperatures, oxidation and hydrolysis occur to a minor extent, which may be acceptable and even improve the product. The most interesting results are obtained with green Java coffee beans. Extraction with superheated water at ca 200°C produces a brown liquid, which has the aroma of coffee. After extraction the beans are whole and homogeneously dark brown: water appears to have permeated throughout the individual beans, they had expanded to about double their initial size. Analysis by head-space gas chromatography shows that the 'coffee' obtained has a much more concentrated extract, but the components

and their proportions are very similar to those found in conventional coffee. Thus a process similar to roasting is occurring for the green beans. These experiments may give rise to a process for producing coffee flavours for the food industry.

Finally, superheated water has also been used to destroy waste by the wet-oxidation process in which toxic organic waste is oxidised to benign compounds using superheated water and air or oxygen.12 Recrystallisations and a wide range of chemical reactions, including hydrolysis, are potential future applications.13

Conclusions

Progress is being made in using the clean solvents carbon dioxide and water to replace conventional organic solvents. In the case of carbon dioxide, the process is well advanced and large industrial processes are functioning. In the case of superheated water, it is still at the research stage.

References

1. C. Cagniard de la Tour, em>Ann. Chim. Phys., 1822, 22, 127. 2. R. S. Oakes et al, Chem. Commun., 1999, 247. 3. L. Haar, J. S. Gallagher and G. S. Kell, NB S/NR C Steam tables. Washington:

Hemisphere, 1984. 4. A. J-M. Lagadec et al, US Patent 6001256, 1999. 5. S. B. Hawthorne, Y. Yang and D. J. Miller, Anal. Chem., 1994, 66, 2912. 6. Y. Yang et al, Anal. Chem., 1995, 67, 4571. 7. A. J-M. Lagadec et al, Environ. Sci. Technol., 2000, 34, 1542. 8. P. J. Hammond, UK Patent 2343890A, 2000. 9. A. Basile, M. M. Jimenez-Carmona and A. A. Clifford, J. Agric. Food Chem., 1998, 46,

5205. 10. Y. Yang, D. J. Miller and S. B. Hawthorne, J. Chem. Eng. Data, 1997, 42, 908. 11. S. B. Hawthorne et al, Environ. Sci. Technol., 2000, 34, 3224. 12. F. J. Zimmernam, US Patent 2665249, 1954.

M. Siskin and A. R. Katritzky, Am. Chem. Soc., 217, 1999.