Immunomodulatory activity of Bhallatak

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Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 1 1. Introduction Immunomodulation is one of the most rapidly developing areas of medical science & disease prevention research and has great promises with regard to the prevention and treatment of a wide range of disorders such as the inflammatory diseases of skin, respiratory disorder and various infectious diseases are disorders caused by organisms such as bacteria, viruses, fungi or parasites. And worlds 25.9% of the death occurred due to the infectious diseases. 1 In addition, infectious diseases are now primarily considered immunological disorders. Immunomodulators are natural or synthetic substances that help regulate or normalize the immune system. Immunomodulators correct immune systems that are imbalance. This elaborate defence system can keep health problems ranging from HIV/AIDS to the common cold at bay. Research on immunomodulation by natural products or synthetic derivatives is of key interest for immunomodulation therapy for a number of reasons. Many plant remedies well-known in traditional medicine or refined natural products in clinical use by directly affecting the pathogen. At least part of their effect is indirect, by stimulating natural and adaptive defence mechanisms of the host. These findings have now given many empirical therapies a rationale, scientific basis and thereby a means for ‘intelligent’ improvement. In discovering the molecular mechanisms by which known remedies exert their effects, chosen elements further down the ‘chain of command’ might be synthesized and applied directly for more rapid and selective cure, omitting unwanted side effects. The direct use of recombinant cytokines, often in combination with antibiotics, is one consequence of this rationale. As herbal immunodulatory drugs has the potential to achieve the greater efficacious drugs for the prevention of infectious diseases. Ayurveda antiquity developed certain dietary and therapeutic measures to arrest/delay ageing and to rejuvenate whole functional dynamics of the body system. This revitalisation and rejuvenation is known as the rasayan chikitsa (rejuvenation therapy) which, in the current context, can be equated to immunomodulation or adaptogenic activity. Traditionally, rasayana drugs are used against a plethora of seemingly diverse disorders with no pathophysiological connections according to modern medicine. Although this group of plants generally possesses strong immunomodulatory activity, only some have been investigated in detail. In this study, apart from an insight into the role of Ayurveda and rasayana in the modulation of the immune system, immunomodulatory activities of other medicinal drugs, along with some compounds isolated from plants are reviewed. Ayurveda physicians believe in preventive therapy rather than curative and the rasayanas are the disease-preventive agents of Ayurveda. 2

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Immunomodulatory activity of Bhallatak

Transcript of Immunomodulatory activity of Bhallatak

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 1

    1. Introduction

    Immunomodulation is one of the most rapidly developing areas of medical science & disease

    prevention research and has great promises with regard to the prevention and treatment of a

    wide range of disorders such as the inflammatory diseases of skin, respiratory disorder and

    various infectious diseases are disorders caused by organisms such as bacteria, viruses, fungi

    or parasites. And worlds 25.9% of the death occurred due to the infectious diseases.1 In

    addition, infectious diseases are now primarily considered immunological disorders.

    Immunomodulators are natural or synthetic substances that help regulate or normalize the

    immune system. Immunomodulators correct immune systems that are imbalance. This

    elaborate defence system can keep health problems ranging from HIV/AIDS to the common

    cold at bay. Research on immunomodulation by natural products or synthetic derivatives is of

    key interest for immunomodulation therapy for a number of reasons. Many plant remedies

    well-known in traditional medicine or refined natural products in clinical use by directly

    affecting the pathogen. At least part of their effect is indirect, by stimulating natural and

    adaptive defence mechanisms of the host. These findings have now given many empirical

    therapies a rationale, scientific basis and thereby a means for intelligent improvement. In

    discovering the molecular mechanisms by which known remedies exert their effects, chosen

    elements further down the chain of command might be synthesized and applied directly for

    more rapid and selective cure, omitting unwanted side effects. The direct use of recombinant

    cytokines, often in combination with antibiotics, is one consequence of this rationale.

    As herbal immunodulatory drugs has the potential to achieve the greater efficacious drugs for

    the prevention of infectious diseases.

    Ayurveda antiquity developed certain dietary and therapeutic measures to arrest/delay

    ageing and to rejuvenate whole functional dynamics of the body system. This revitalisation and

    rejuvenation is known as the rasayan chikitsa (rejuvenation therapy) which, in the current

    context, can be equated to immunomodulation or adaptogenic activity. Traditionally, rasayana

    drugs are used against a plethora of seemingly diverse disorders with no pathophysiological

    connections according to modern medicine. Although this group of plants generally possesses

    strong immunomodulatory activity, only some have been investigated in detail. In this study,

    apart from an insight into the role of Ayurveda and rasayana in the modulation of the immune

    system, immunomodulatory activities of other medicinal drugs, along with some compounds

    isolated from plants are reviewed. Ayurveda physicians believe in preventive therapy rather

    than curative and the rasayanas are the disease-preventive agents of Ayurveda.2

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 2

    Semecarpus anacardium (SA) Linn. (Family Anacardiaceae), commonly called as

    Bhallatak, It is well-known for its Rasayana properties in Ayurveda. Immunomodulation using

    SA provide an alternative to conventional chemotherapy for a variety of diseases, especially

    when host defence mechanism has to be activated under the conditions of impaired immune

    response or when a selective immunosuppression is desired in situations such as autoimmune

    disorders.3 It appears that the normal way by which the immune system works is through its

    own modulation by factors usually synthesised by the immune cells.4

    SA has been shown; the nut oil from Semecarpus anacardium (SA) was shown to be

    cytotoxic to human leukaemic cell lines.5 The ethyl acetate extract contains a biflavonoid

    known as tetrahydroamentoflavone (THA) that inhibited the enzyme cyclooxygenase-2 (COX-

    2).6 The alcoholic extract of dry nuts had anti-fungal activity while of nut shells were shown to

    prevent lipid peroxidation.7 The nut milk extract is effective in restoring the fragility of

    lysosomal membranes in aflatoxin-induced hepatocellular carcinoma.8 The immune response

    requires timely interplay of multiple cell types within specific microenvironments to maintain

    immune homeostasis. The selectivity and flexibility that is necessary to regulate cell traffic

    under homeostatic and diseased conditions are provided by the differential distribution and

    regulated expression of cytokines and their receptors. As a consequence, cytokines are

    responsible for the development of phenotypes and are, therefore, logical targets for therapeutic

    immune modulation.9

    1.1 Classification of immunomodulation

    In clinical perspectives, immunomodulators can be classified into three categories.

    Immunoadjuvants are used for enhancing the efficacy of vaccines and therefore could be

    considered to be specific immune stimulants. Adjuvants such as monophosphoryl lipid A,

    immunostimulating complex (ISCOM)10

    Immunostimulants are compounds leading predominantly to a non-specific stimulation of

    the immune system and are also called mitogens. These agents, generally, interact not just

    with one but also with other types of immune competent cells, because of the close link between

    the non-specific and specific immune system. This is one handicap in developing effective

    immunostimulating agents without any side-effects, since in some cases immunostimulants

    may also stimulate T-suppressor cells, and thereby reduce the immune resistance. The terms

    immunomodulators or immunoregulators therefore very often seem to be more appropriate.11

    Immunosuppressants are agents that could be used for control of pathological immune

    response in autoimmune diseases, graft rejection, graftversus- host disease, hypersensitivity

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    immune reaction (immediate or delayed type) and immune pathology associated with

    infections.12

    1.2 Immunomodulators in Ayurveda

    A significant part of Ayurvedic therapeutics is preventive in nature. The capacity of the body

    to resist disease, is stimulated. The rasayanas of Ayurveda are believed to be

    immunomodulators in modern terminology. Ayurveda considers that an individual, when

    advanced in age, accumulates wastes and toxic substances in his/her cellular system, which

    disrupt the normal metabolism, leading to loss of immunity, causing disorders or diseases and

    finally hastening the ageing process. Ayurvedic experts have developed various methods to

    detoxify the tissues and rejuvenate the body by a special treatment regimen called rasayana

    chikitsa. The immune system is known to be involved in the aetiology of many diseases as well

    as in the pathophysiological mechanisms.

    Ayurveda emphasises the promotion of health, a concept of strengthening host defences

    against different, so rasayana drugs are particularly recommended for the treatment of immune

    disorders. The development of agents capable of moving the patients immune system from a

    state of deficiency to one of more normal function would be likely to have a significant impact

    on disease. Such agents would not be a cure but would control the manifestation and course of

    disease some plants and their constituent compounds are claimed to induce para immunity, the

    non-specific immunomodulation of macrophages, granulocytes, natural killer cells and

    lymphocytes and complement functions13. Many studies have been undertaken to provide

    scientific support for the use of rasayana drugs as immunomodulators and adaptogens.

    Rasayana drugs have been reported to treat generalised weakness 14and to afford protection

    from cyclophosphamide-induced leukopenia.15

    1.3 Concept of 'Vyadhirodhak Chamatav'.

    There is a difference in the concept of bodys resistance to disease in traditional Indian

    system of medicine, According to Ayurvedic theory a harmonious balance between

    three humors of the body viz. Vayu', Pitta and Kaph is needed for positive health; imbalance

    of these may cause disease(s). A significant part of Ayurvedic therapeutics is preventive in

    nature. It aims to promote positive health so that individuals do not suffer from disease. This

    is the concept of "Vyadhirodhak chamatav". ie capacity of the body to resist disease.

    Obviously, the immune system. As recognised in modern biology which provides protection

    against microbes, should be a part of it. An entire section of the Materia Medica of Ayurveda

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    termed Rasaynas' is devoted to enhancement of body's resistance. Interestingly the

    prescribed this section include not only procedures under drugs ('Aushadhi') but also "Aachar"

    (daily routine including exercise), "Aahar" (diet and nutrition) and "Vyavhar (mental attitude

    and discipline) which are equally important in achieving the desired goal. In comparison to this

    concept of Rasaynas' in Ayurveda.16

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    2. Literature Review

    2.1 Classical Text

    2.1.1 Charak samhita:

    According to charak samhita, chikitsasthanam, the sneha of bhallatak is taken into one vessel

    along with godugdha mulethi is & cooked with the oil. The same process repeated for 100

    times. The uses are generally similar to that of bhallatakksoudhra

    2.1.2 Susruta samhita:

    In suhruta Samhita, properties and uses of bhallatak oil mentioned Bhallatak oil uses are is

    bakarak, balya

    2.1.3 Shankar nighantu:

    In Shankar nighantu, different synonyms like bhallatak ,bhilawa, bhela, marking

    nut,malaccabean, and properties like astringent, hot ,sukarjanan, madhur , and light it is used

    in vatalkapha, udarroga, kustha, babascer, sangrahniya, gulam jawara, agniga, mandya,

    krimirog and varann The friuts of ballatak sweet, when ripe, light,astringent, snigdha, tikana,

    hot, chedan, bhedan and appetizer. The bark of fruit is sweet, light, astringent, and it is used in

    deepan, pachan, uderrog, swelling and fever.

    Shodan of ballatak: Keep the ripe fruits of ballatak in water, fruits that are sink into the water,

    that take for the shodahan with the same quantity of water, then rubbed on the brick powder.

    2.1.4 Bhav prakash nighantu:

    It is a 22-40 feet long tree bark.1 inch thick and grayish in colour. Flowers are inflorescence

    yellowish in colour. Fruits are l inch long, black in colour. Different properties, chemical

    composition and there therapeutics uses and shodahan of bhallatak mentioned in Bhavpraksh

    nighantu.

    2.1.5 Nighantu Adarsh:

    Bhallatak, aruskar, agnimukh, bhilawa, biladur, veervariksha. It is astringent, sweet, hot, bitter

    and kaphavata nashka. It is a medium size tree. Found in Assam, madhyabharat and

    dakshinabharat. The leafs are long, ovate, Flowers are yellow in colour.The fruits are heart in

    shape, black and white in colour when unripe'

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    2.1.6 Raj nighantu:

    Bhallatak, agni, dahan, patan, aruskar, anal, krimighan tailbeej, vaatari, sufoot bijak,

    prithakbeej, dhanuurbeej, beejeepadak,etc. Sixteen synonyms are given. It is katu, tikta, kasaya

    in rasa. It is used in vata disorders, udarvikar, anaah and parmeha. The fruits of bhallatak are

    madhur rasa in and ushna. It is used in kapha, sarram, swasarog, annah, vibandsool, udarrog

    and kriminashak

    2.1.7 Dhanvantari nighantu:

    Aruskar, dahan, tapan, agnik, bhallatak, agnimukha, virtaru and dhanu. It is katu tikta, madhur,

    ushna virya, krimighan and vata, pitta nashak

    2.1.8 Priya nighantu:

    Bhallatak is sharp like bhala weapon. It cause irritation when touch the fruit without precaution.

    It is used in kusta, arsha, kapharog, raktadustijanya vikar, agnimandya, krimiroga.

    2.1.9 Dravyaguna Vijnana:

    Botanical description: A moderated size deciduous tree, exudation a dark juice. Young

    branches inflorescence petiole and under side of leafs pubescent. Leaves- oblong, ovate

    rounded at apex, cartilaginous at margin, very coriaceous. Flowers- fasciculate, arranged in

    erect, compound greenish yellow colour. Fruits drupes, obliquely oval or long, smooth, shining,

    purplish-black when ripe, cup orange. Flowering round the year, mostly during May-June,

    fruits ripe from November to February.17

    2.1.10 Ayurvedic pharmacology:

    Class: Kushthaghna, Deepaniya, Mootrasangrahaniya, Nyagrodhadi, Mustadi

    Kula: Amra kula

    Family: Anacardiacee (Ana-like, Cardium-heart)

    Method of purification: Take out the bhallataka fruit along with the stalk and keep it in the

    powder of bricks for period of one week. Clean and wash thouroughly by a rubbing it, then

    boil it with milk. This purifies bhallataka impure bhallataka act as a toxin, hence it should be

    used without purifies. Small children, pregnant not women, older people, persons having

    pittaprakruti, patients having tendency to bleed, and those who are allergic to Bhallatak should

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 7

    not take bhallataka. It is important for a vaidya to know the contraindication rather than the

    indication of bhallataka18

    2.1.11 Ayurvedic pharmacopeia of India:

    Bhallataka consists of mature fruit of Semecarpus anacardium Linn. (Fam.Anacardiaceae), a

    medium sized tree found in moist deciduous forests all over the country.

    Synonyms

    Sanskrit : Bhallatakam

    Assamese : Bhelaguti

    Bengali : Bhela

    English : Marking Nut

    Gujrati : Bhilam

    Hindi : Bhilawa

    Kannada : Bhallataka

    Malayalam : Chera

    Marathi : Bibba

    Oriya : Bhollataki, Bholai

    Punjabi : Bhilawa

    Tamil : Tatamkottai, Scramkotati

    Telugu : Nallajidi, Nallajidiginga

    Urdu : Baladur, Bhilavan

    Description

    Macroscopic: Fruit laterally flattened, drupaceous, dark brown, nut 2.5-3 cm long, obliquely

    ovoid, smooth, shining with residual receptacle.

    Microscopic:

    Fruit - Pericarp differentiated into epicarp, mesocarp and endocarp; in longitudinal section

    pericarp shows outer epicarp consisting of single layer of epidermal cells which are elongated

    radially and lignified, characteristic glands found in pericarp which exude oil globules and arise

    as small protuberances in epicarp and due to pressure exerted by cells of mesocarp, some of

    epidermal cells and cuticle rupture and oil globules exude from oil glands; mesocarp a very

    broad zone, 30-40 layers thick, composed mostly of parenchymatous cells having lysigenous

    cavities and fibro-vascular bundles, below epidermis a few outer cells of parenchyma smaller

    as compared to rest; rosette crystals of calcium oxalate found scattered in parenchymatous

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 8

    cells, some cells get dissolved and form lysigenous cavities which increase in size with maturity

    of fruit, cavities do not have any special lining and contain an acrid and irritant yellowish oily

    secretion; endocarp consists of two distinct layers, innermost prismatic, very much elongated

    radial walls, being highly thickened, outer layer shorter and thinner than prismatic layer but

    cells similar to the former; number of mesocarp parenchyma contain rosette crystals of calcium

    oxalate and oil drops in oil glands; lysigenous cavities of mesocarp contain oily vesicating

    substance, insoluble in water and soluble in alcohol, ether, chloroform. Powder - Dark-brown;

    shows rosette crystals of calcium oxalate and oil globules.

    Identity, Purity and Strength

    Foreign matter : Not more than 1 %

    Total Ash : Not more than 4 %

    Acid-insoluble ash : Not more than 0.5 %

    Alcohol-soluble extractive : Not less than 11 %

    Water-soluble extractive : Not less than 5 %

    Constituents: A Tarry Oil containing Anacardic Acid, Non-Volatile Alcohol (Cardol).

    Properties and action

    Rasa : Madhura, Katu, Tikta, Kasaya

    Guna : Laghu, Snigdha,Tiksna

    Virya : Usna

    Vipaka : Madhura

    Karma : Dipana, Kaphahara, Pacana, Vatahara, Chedi, Bhedi, Medhya

    Important formulations: Amrta Bhallataka Leha, Bhallataka

    Rasayana, Bhallatakadi Modaka

    Therapeutic uses: Arsa, Anaha, Grahani, Gulma, Krimi, Kustha.

    Dose: 1.2 g. of the drug in Ksirapaka form.19

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 9

    2.2 Reported pharmacological activities of Semecarpus anacardium.

    Table 1: Reported pharmacological activities of SA

    S.

    No

    .

    Extract/

    Formula

    tion

    Pharmacological

    activity reported

    In vivo

    models

    (Humans/An

    imals)

    In vitro

    models (cell

    lines/

    chemical or

    microbial

    assay)

    Dose Ref

    1 Nut

    extract

    Breast cancer - T47D cell

    line

    400

    mg/mL

    20

    2 Seeds

    (Biflavan

    oids)

    COX inhibitors Male spargue

    Dawley rats

    (Rat paw

    edema assay)

    - 100 g/kg 21

    3 Nut milk

    extract

    Antidiabetic Male albino

    wistar rats

    - 300 mg/kg

    b.wt. (For

    21 days)

    22

    4 Nut

    extract

    Antiarthritic Male albino

    wistar rats

    - 150 mg/kg 23

    5 Nut milk

    extract

    Antiarthritic Male albino

    wistar rats

    - 150 mg/kg 24

    6 Nut

    extract

    Immunomodulato

    ry and

    Antiarthritic

    Humans - 1g/mL 25

    7 Nut milk

    extract

    Antiarthritic Male albino

    wistar rats

    (Freunds adjuvant

    induced

    arthritis)

    - 150 mg/kg 26

    8 Chlorofo

    rm

    extract

    of SA

    Aphrodisiac Male albino

    wistar rats

    - 150 and

    300 mg/kg

    27

    9 Ethanol,

    acetone

    and

    aqueous

    extract

    Antioxidant - DPPH and

    ABTS assay

    100

    g/mL

    (DPPH

    assay) and

    0.01 to 0.5

    mg/mL (

    ABTS

    assay)

    28

    10 Nut milk

    extract

    Antidiabetic (type

    II)

    Male spargue

    Dawley rats

    (Streptozotoc

    in induced

    - 200 mg/kg 29

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 10

    diabetes

    mellitus)

    11 Fruit

    extract

    Hepatoprotective Male albino

    wistar rats

    - 250 and

    500

    mg/kg

    30

    12 Methano

    lic

    extract

    Anti fungal - Fungal

    strains

    ( Fusarium

    oxysporum,

    Rhizctonia

    solanii,

    Alternaria

    spp., and

    Sclerotium

    rolfsii)

    6.25,12.5,

    25, 37.5,

    50 and

    62.5

    g/mL)

    40

    13

    Petroleu

    m ether

    nut

    extract

    Antibacterial - Microorganis

    m

    (E.Coli,Bacill

    us

    subtilis,Micro

    coccus

    luteus,Klebsi

    ella

    pneumonia,

    Streptococcu

    s

    aureus,Proteu

    s vulgaris,

    Salmonella

    typhi)

    150 l

    40

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 11

    14 Aqueous

    and

    organic

    extract

    Antimicrobial - Microorganis

    m

    (Staphylococ

    cus aureus,

    Shigella

    flexneri,

    Bacillus

    licheniformis,

    Vibiro

    cholera,Pseud

    omonas

    aeruginosa,

    Streptococcu

    s aureus,

    Bacillus

    brevis.)

    10,50,100

    mg/ml

    10mg

    G/ml

    31

    Ayurvedic formulation.

    15 Amrut

    Bhallata

    kavaleha

    (Electuar

    y)

    General tonic and

    vitalizer

    Humans - 1 to 2

    teaspoonfu

    l for 2

    times

    32

    16 Bhallata

    kasava

    (Wine)

    Neuralgia and

    asthma

    Humans - 2 to 4

    teaspoonfu

    l for 2

    times

    41

    17 Suran

    vatak

    (Pills)

    Piles and

    anorectal diseases

    Humans - 2 pills

    (500 mg

    pill) for 2

    times

    41

    18 Sanjeeva

    ni Vati

    (Pills)

    Dysentry and

    diarrhea

    Humans - 2 pills

    (250 mg

    pill) for 3

    times

    41

    19 Bhallata

    k Parpati

    (Powder)

    Rheumatic

    diseases

    Humans - 250 mg

    for 3 times

    41

    20 Narsimh

    a

    Choorna

    (Powder)

    General

    restorative

    Humans - 1 to 2 gm

    for 2 times

    41

    2.3 Various Antidotes of SA Toxicity

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    Cows milk, Cows Urine is recommended as the antidote for bhallataka blisters. Neem is used

    as topical Antidotes.33

    2.4 Drug interaction of SA

    2.4.1 Manodeep Chakraborty et al., 2010 have reported Interaction of Semecarpus

    anacardium L. with propranolol against isoproterenol induced myocardial damage in

    rats

    With a view to evaluate the cardio protective effect of ethanolic extract of S. anacardium nut

    and the possible interaction with propranolol against isoproterenol induced myocardial damage

    in rats, female Sprague-Dawley rats were pre-treated with propranolol (10 mg/kg for 7 days),

    low and high doses of S. anacardium (100 and 500 mg/kg for 21 days) and their combination

    orally and subsequently subjected to isoproterenol administration (150 mg/kg, sc) for two

    consecutive days. The influence of prophylactic treatment was analysed by quantification of

    biomarkers and antioxidants, electrocardiographic parameters and histopathological

    observations. The activities of lactate dehydrogenase and creatinine phosphokinase-MB were

    reduced in serum and raised in heart tissue with concurrent elevation in superoxide dismutase

    and catalase activities as well as reduction in thiobarbituric acid reactive species levels

    significantly in all treated groups compared to isoproterenol group. Similarly,

    electrocardiographic changes were restored to normalcy in all treated groups. To conclude,

    combination of high dose of S. anacardium with propranolol was found to be most effective in

    alleviating the abnormal conditions induced by isoproterenol.

    This study was to elucidate the role of S. anacardium nut extract during

    myocardial dysfunction and metabolic derangement induced by isoproterenol in rat heart and

    also to explore its pharmacodynamics interaction with conventional cardio protective drug.

    Propranolol. The results revealed the beneficial role of S. anacardium when treated

    concurrently with propranolol in conditions of anticipated cardiac injury.

    Results it may be concluded that the SANE both at low (100 mg/kg) and high doses (500

    mg/kg) possess cardio protective efficacy when given prophylactically in experimental

    animals. Moreover, combined therapy of SANE and PRO demonstrated synergistic cardio

    protective potential than when they were used alone. The combination of high dose of SANE

    and PRO was found to have best effect. However, combined therapy of SANE and PRO must

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    be further refined for adjustment of doses as higher doses of SANE and PRO may pose

    negative implication due to excessive pharmacological effects.

    2.5 Adverse effects of bhallataka:

    Impure or pure bhallataka if taken in excess dose, cause pruritus and burning sensation of anus

    and tip of the penis, excessive perspiration, thirst and reduced red dusky urination, in such

    condition, one should or stop taking bhallataka and should start pitta alleviating drug like

    doorvadigana, sarivadigana. In condition like burning sensation or pruritus and swelling of

    skin, sesamum oil, coconut oil, resin ointment should be applied locally: In 3-4 ghee or days

    the above symptoms subside. The above should be kept handy before starting Bhallatak

    treatment.34

    2.6 Safety profile:

    The drug should be used with care, preferably under the direction of a qualified practioner,

    since the Anacardic acid are allergenic. The maximum tolerated dose of a 50% alcoholic extract

    of the fruit when given interaperitoneally to mice was found to be 250/mg/kg body weight.35

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 14

    3. Phytochemical profile of Semecarpus Anacardium.

    SA can be greatly aided by the isolation of its active principle and determination of structure

    and function relationship. Based on this principle a lot of phyto pharmaceuticals from different

    parts of S. anacardium have been isolated. Phytochemical examination revealed 3.85% of total

    ash, 0.33 % of acid insoluble ash, 11.27 % alcohol soluble extractive, 11.84% water soluble In

    alternative medicine, medicinal plant preparations have found wide spread use particularly in

    the case of diseases not amenable to treatment by modern methods. A variety of nut extract

    preparations from S. anacardium are effective against many diseases viz. arthiritis, tumours,

    infections etc.36 Various anacardoflavanone have been isolated from the nut shells and

    characterized isolated one more biflavonoid namely tetrahydrorobustaflavone from the

    defatted nuts of the S. anacardium and structure characterized. The leaves of the S. anacardium

    found to contain amentoflavone as the sole compound. 37The corrosive juice from the pericarp

    of the fruit found to contain catechol, fixed oil and anacardol (C18H13O3.COOH) to which the

    corrosive properties of the juice are due to two phenolic acids C16H15O3.COOH and

    C14H13O3.COOH. 38 From the seeds of S. anacardium, a new phenolic glucoside, anacardoside,

    was isolated, and its structure and configuration were elucidated by a combination of NMR

    techniques as-l-O- -D-glucopyranosyl- (1 - 6) - -D-glucopyranosyloxy-3- hydroxy-5-

    methylbenzene.39

    Table 2: Qualitative Phytochemical Analysis of Various Extracts of Semecarpus

    anacardium Leaves.

    S. No.

    Name of the Test

    Procedure

    Observation Soxhlet Extracts of Leaves

    1. Alkaloids A B C D E F G H I J K L M N

    i Dragendo

    rffs Test Orange Red

    ppt.

    - - - + - - - - - - - + - -

    ii Mayers Test

    Whitish

    Yellow or

    Cream

    coloured

    ppt.

    - - - + + - - + - - - - + -

    iii Hagers Test

    Yellow

    coloured

    ppt.

    - - - - - - - + - - - + + -

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 15

    iv Wagners Test

    Reddish

    Brown ppt.

    - - + + - + + + - - + + + +

    2. Saponins A B C D E F G H I J K L M N

    i Foam

    Test

    Foam

    persists for

    10mins.

    + - - + + - - - + - - - + -

    3. Carbohydrates A B C D E F G H I J K L M N

    i Molisch

    Test

    Purple or

    reddish

    violet color.

    - - - - - - - - - - - + - -

    ii Fehlings Test

    Brick Red

    ppt.

    - - - - - - - - - - - - + -

    iii Benedicts Test

    Red ppt. - - - - - - - - - - - - + -

    4. Glycosides A B C D E F G H I J K L M N

    i Legals Test

    Pink to Red

    color.

    + - + + + + - - - - + - + -

    ii Baljet

    Test

    Yellow to

    Orange

    color.

    + + - + - - + + + + - + - -

    5. Tannins A B C D E F G H I J K L M N

    i Lead

    acetate

    Test

    White ppt. - - - - - - - - - - - - - -

    ii Ferric

    chloride

    Test

    Dark Blue

    or Greenish

    Black.

    - - - - - - - + - - - - - -

    iii Potassium

    dichromat

    e Test

    Yellow

    color ppt.

    - + + - - - + + + + - - - -

    iv Gelatin

    Test

    White ppt. - - + + + - - + + + - + + -

    v Potassium

    ferric

    cyanide

    Test

    Deep red

    color.

    - - - - - - - - - - - - - -

    6. Flavonoids A B C D E F G H I J K L M N

    i Shinodas Test

    Cherry Red

    color.

    - - - - + - - + - - - + - -

    ii Alkaline

    Reagent

    NaOH

    Test

    Intense

    Yellow

    color.

    - + - + + - + + - + - + + +

    iii H2SO

    4

    Test

    Yellow or

    Orange

    color.

    - + + + + - + + + + + + - -

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 16

    iv Lead

    acetate

    Test

    Yellow

    color ppt.

    + + - + - - - + + - - + + +

    7. Steroids A B C D E F G H I J K L M N

    i Salkowsk

    i Test

    Bluish red

    to cherry

    color in

    Chloroform

    layer &

    Green in

    acid layer.

    - + - - - - - + - - + + - +

    ii Liberman

    n

    burchard

    Test

    Brown ring

    at junction

    & green or

    deep red

    upper layer.

    - + - + - - + - + + + + - +

    8. Phenols A B C D E F G H I J K L M N

    i Ferric

    chloride

    Test

    Bluish

    Black color.

    - - - - - - - + - - - - - -

    9. Proteins A B C D E F G H I J K L M N

    i Biuret

    Test

    Pinkish or

    Purple

    violet color.

    - - - - - - - - - - - - - -

    ii Ninhydrin

    Test

    Blue color. - - - - - - - - - - - - - -

    iii Xanthopr

    oteic Test

    Orange

    color.

    - - + + - - - + - + - + + -

    10. Monosaccharide A B C D E F G H I J K L M N

    i Barfoeds Test

    Red ppt. - - - - - - + - - - - - - -

    11. Hexose Sugars A B C D E F G H I J K L M N

    i Selwinoff

    s Test for ketohexos

    e like

    fructose

    Red color. + - - + - - - + + + - + - -

    ii Tollens phloroglu

    cinol Test

    for

    galactose

    Yellow to

    Red color.

    + + + + + + + + + + + + + -

    iii Cobalt

    chloride

    Test

    Upper layer

    Greenish

    blue &

    + + + + + + + + + + + + + +

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 17

    Lower

    Purplish.

    12. Diterpenes A B C D E F G H I J K L M N

    i Copper

    acetate

    Test

    Emerald

    Green

    color.

    + + - - + - + + + + + + - +

    13. Nonreducing

    Polsaccharides [Starch]

    A B C D E F G H I J K L M N

    i Iodine

    Test

    Blue color. - - - - - - - + - - - - - -

    ii Tannic

    acid Test

    ppt

    formation.

    - - - - + - - + - - - - - -

    14. Mucilages & Gums A B C D E F G H I J K L M N

    i Rutheniu

    m Red

    Test

    Pink color. + + - - - - + + + + - - - -

    (+ ) = indicates presence, ( - ) = indicates absence.

    A= water, B= chloroform, C= toluene, D= carbon tetrachloride, E= ethyl acetate, F= hexane,

    G= ethyl alcohol, H= methanol, I= acetone, J= 2-propanol, K= petroleum ether 60-80 C, L=

    2-butanone, M= dichloromethane, N= ethyl ether.40

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 18

    4. Shodhana of SA (Purification)

    Bhallataka (SA) is reported under upavisha dravya (semi poisonous drugs), in classical

    Ayurvedic pharmacopoeias. It is advocated that shodhana (Purificatory procedures) of the

    fruits should be carried out before its internal administration. Though there are different

    shodhana methods mentioned in Ayurveda, Ayurvedic Pharmacopoeia of India (API)

    recommends only one method for the shodhana of Bhallataka fruits. In study, cows urine,

    cows milk and brick powder, were used as media. Ayurveda advocates bhallataka after

    shodhana (purificatory procedures).Though there are different shodhana methods mentioned

    in Ayurveda41

    4.1 Effects of Non-Purified (SA) and effect of liquid media in shodhana

    In Ayurvedic literature, the synonym Sopha hetu, Spota hetu, agnika are given to this drug

    based on its blister causing nature. The oil in the fruit is responsible for the irritation. The

    bhallataka fruit contains 90% Anacardic acid and 10% of Cardol. Other chemical constituents

    are bhilawanol , semecarpol and anacardol. Recent studies reported that bhilawanols are known

    as urushiols. Anacardic acids are closely related to urushiol. Another study reported that the

    corrosive juice from the pericarp of the fruit is found to contain catechol, fixed oil and

    anacardol (C18H13O3.COOH) to which the corrosive properties of the juice are due to two

    phenolic acids C16H15O3.COOH and C14H13O3.COOH. The media gomutra (cows urine) is

    reported for its antimicrobial, antibacterial etc. Cows milk is recommended as one of the

    antidote for bhallataka blisters. Brick powder is having adsorbent property; by which it absorbs

    irritant oil in the fruit.42

    4.3 Method of Sodhana

    Bhallataka fruits, sunken in water, were randomly taken. The thalamus portion of the fruits

    was removed with the help of a steel cutter. Then it was taken in a vessel containing gomutra

    (cows urine) and kept for seven days. Every day the fruits were taken out of the media and

    washed with water and fresh gomutra was used. On eighth day Bhallataka was washed and

    shifted to the vessel containing godugdha (cows milk) and kept for seven days. Each day it

    was washed with water and fresh Godugha was added. On 15th day the samples were taken

    out of the media and washed with water then shifted to a bag containing brick powder and

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 19

    rubbed thoroughly. It was allowed for three days in the bag containing brick powder. On 18th

    day it was washed thoroughly with hot water to remove the brick powder in the sample. Later,

    the samples were dried properly to remove the moisture and stored in air tight glass container

    for further studies. The same shodhana procedure was repeated thrice to standardize the

    procedure pharmaceutically.

    4.4 Impact on Anacardol Content.

    Due to the decorboxylation of the oil, the anacardic acid gets converted into less toxic

    anacardol. Decorboxylation process may start right from cutting the fruit itself and will be

    catalysed by giving heat/fire Treatment.43 The increased level of anacardol in the shodhita

    bhallataka may be due to the decorboxylation of the anacardic acid in the fruits. More

    percentage of oil might have got reduced by soaking the fruits in the gomutra and godugda.

    The brick powder is having the adsorbing nature, so some percentage of oil may be absorbed

    by the brick powder. There are probable chances that some chemical changes might have taken

    place due to the various Medias like gomutra, godugda etc used for its purification. Further

    studies should be carried out to find out the chemical interactions between the media and the

    bhallataka fruits during shodhana procedure. Anacardol in raw bhallataka was 47.51% and

    50.62% in processed. Shodhana (purificatory procedure) increases the anacardol level in

    shodhita bhallataka fruit samples. More percentage of the anacardol was due to the conversion

    of toxic urushiol into Anacardol.44

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 20

    5. Methods for Testing (SA) Immunological Factors

    The whole animal model is the most classic pharmacological screening model, which is very

    important at the aspect of medicine evaluation because it can apparently respond to the efficacy,

    side effect and toxicity of medicines in whole. Although this method is high cost and low

    efficient, at present it is still a primary way to drug discovery and evaluation. Several in vitro,

    in vivo methods of pharmacological screening of medicinal plants having immunomodulatory

    activity have been listed.45

    5.1 In vitro methods:

    1. Inhibition of histamine release from mast cells

    2. Mitogens induced lymphocyte proliferation

    3. Inhibition of T cell proliferation

    4. Chemiluminescence in macrophages

    5. PFC (plaque forming colony) test in vitro

    6. Inhibition of dihydro-orotate dehydrogenase

    5.2 In vivo methods:

    1. Spontaneous autoimmune diseases in animals

    2. Acute systemic anaphylaxis in rats

    3. Anti-anaphylactic activity (Schultz-Dale reaction)

    4. Passive cutaneous anaphylaxis

    5. Arthus type immediate hypersensitivity

    6. Delayed type hypersensitivity

    7. Reversed passive arthus reaction

    8. Adjuvant arthritis in rats

    9. Collagen type II induced arthritis in rats

    10. Proteoglycan-induced progressive polyarthritis in mice

    11. Experimental autoimmune thyroiditis

    12. Coxsackievirus B3-induced myocarditis

    13. Porcine cardiac myosin-induced autoimmune myocarditis in rats

    14. Experimental allergic encephalomyelitis

    15. Acute graft versus host disease (GVHD) in rats

    16. Influence on SLE-like disorder in MRL/lpr mice

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 21

    6. Experimental Protocol to evaluate (SA) immunomodulatory activity.

    Determination of the immunomodulators effects of SA extracts in adjuvant induced arthritic

    (AIA) rat model. AIA is an erosive autoimmune polyarthritis involving both humoral and cell

    mediated immune responses that resemble human rheumatoid arthritis (RA). At cellular level

    immunosuppression occurred during the early phase of the disease. There was mild synovial

    hyperplasia and infiltration of few mononuclear cells in SA treated animals. The induction of

    nitric oxide synthase (NOS) was significantly decreased in treated animals as compared to

    controls. These observations suggest that the herbal extracts caused immunosuppression in

    AIA rats, indicating that they may provide an alternative approach to the treatment of arthritis.

    6.1 Adjuvant arthritis in Animal (Rats).

    6.1.1 Animal Model.

    Both male and female albino rats (Sprauge Dawley Strain) 10 rats taken 5 male 5

    female.

    6.1.2 Development of arthritis.

    Rats were injected with 300 l of Complete Freunds Adjuvant (CFA) in the right hind footpad

    and left overnight for development of inammation.

    6.1.3 Evaluation of arthritis.

    Measuring the thickness of inamed ossicular tissue using a dial gauge calliper assessed degree

    of arthritis. Severity of inammation was classied using a six-point scale based on

    enlargement, erythema and edema of the tissue.

    6.1.4. Treatment

    To determine the optimum dose, animals were initially treated with 200, 100, 50, 25, 12.5, and

    200 mg/kg body weight of the SA Extract separately. Intraperitoneal administration the

    according to body weight it should be found to be the optimum dose for immunomodulatory

    property, hence used for further studies. All the animals received SA for 25 days.

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 22

    6.2. Lymphocyte proliferation assay used to determine the Cell mediated immunity.

    Lymphocyte proliferation assay can be performed using mitogen Con-A. Both the treated and

    untreated animals were sacriced to remove the spleen in RPMI-1640 medium. Spleen was

    crushed and cell suspension was washed with plain medium. Cells were lysed with 0.9%

    ammonium chloride and re suspended in complete medium with 10% fetal calf serum. Cells

    were cultured at a nal concentration of 3 105cells/100 l/well in triplicate in at bottom

    microtiter tissue culture plates. The optimal concentration of 0.05 g/ml (in vitro concentration

    titration done earlier) of both the extracts was added to the wells separately and in combination

    with Con-A (5.0 g/ml). After 3 days of incubation at 37 C under humidied air supplemented

    with 5% CO2 , 1 ci Hthymidine was added to each well. Six to eight hours later cells were

    harvested and aspirated on to glass-ber lter papers using NUNC, Automatic Cell Harvester

    and the radiolabel incorporated into DNA was counted using LKB auto beta counter.

    6.3. Histology of joint tissues.

    Rats were sacriced to remove the knee joints. Specimens were xed for 24 h in 2%

    glutaraldehyde in phosphate buffer saline, bisected, decalcied and returned to 2%

    glutaraldehyde and submitted for routine parafn embedding. Tissue sections were stained

    using hematoxylin and eosin stain. The histological ndings were graded on the basis of

    synovial hyperplasia and mononuclear cell inltration.

    6.4. Measurement of induction of NO in activated macrophages ( Estimation of Nitric

    oxide production )

    Macrophage activation assay was performed by injecting rats with thioglycolate

    intraperitoneally 72 h prior to sacrice. On the day of sacrice, cold RPMI-1640 incomplete

    medium was administered intraperitoneally to ush out the activated macrophages. The process

    was repeated 810 times to obtain a good yield of macrophages. Once the activated

    macrophages were recovered, cells were centrifuged and washed thrice with plain medium,

    counted and plated at a nal concentration of 5 105cells/well. Plates were incubated for 4h

    at37C in CO2 incubator. Floating cells were removed and the wells were washed twice with

    warm medium to avoid the leaching of the adhered macrophages. Cells were then treated with

    0.05 g/ml of SA. Culture soup was collected for estimation of NO production by Griess

    Reagent method. Briey, 100 l of cell supernatant mixed with 1% sulfanilamide/0.1%

    naphthylethylenediamine/2.5% H3PO was incubated at room temperature for 10 min to form

    a chromophore. The absorbance was read at 550 nm and NO was measured using NaNO2as

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 23

    standard. Quantitation was done against standard curve generated using known quantities of

    sodium nitrite.46

    6.5. Cell culture

    Mononuclear cells were isolated from heparinized venous blood and synovial uid (SF) by

    density sedimentation method). Briey, blood/SF was diluted in phosphate buffered saline

    (PBS), pH 7.2 and layered carefully on lymphoprep at a ratio of 3:1 and centrifuged at 1800

    rpm for 30 min. Cells in the interface layer were carefully separated, washed with PBS thrice

    and resuspended in RPMI 1640 supplemented with 25 mM HEPES, 2 mM L-glutamine,

    penicillin (100 U/ml), streptomycin (100 _g/ml) and 10%heat inactivated Fetal Bovine Serum

    (FBS). Cell concentration was adjusted as per the requirement of the experiment. Cell viability

    after 18 h was assessed by mitochondria-dependent reduction of a yellow tetrazolium dye 3-

    [4,5-dimethylthiazol-2yl]-2, 5-diphenyltetrazolium bromide to insoluble purple formazan by

    dehydrogenases at the end of 18 h culture. It will showed the Percentage cell viability.

    6.6. Cytokine ELISA

    Cells were treated with LPS (10 ng/ml) in presence and absence of different doses (1.0, 0.5 and

    0.2 mg/ml) of Semecarpus anacardium crude ethanolic extract. The culture was incubated for

    18 h at 37 C, 5% CO and supernatants and cells were harvested for ELISA and RNA extraction,

    respectively. Pro inammatory cytokines TNF-alpa, IL-1beta, IL-6 and IL12p40 in the culture

    supernatants were estimated by commercial sandwich ELISA according to the manufacturers

    instruction. Briey, plates were coated with monoclonal capture antibody by overnight

    incubation at 42C and blocked with 10% FBS in phosphate buffered saline (PBS) for 1 h.

    Samples and recombinant standards were added to the plates and incubated for 2 h. Cytokines

    were detected by addition of horseradish peroxidase conjugated streptavidin labelled

    antibodies. Color was developed using tetramethylbenzidine (TMB) for 15 min and absorbance

    was recorded at 450 nm.47

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 24

    7. Reported immunomodulatory activity of Semecarpus anacardium.

    Table 4. Reported Immunomodulatory activities of Semecarpus anacardium

    S.No Extract :

    E/

    Formulati

    on : F

    Description In vivo models

    or

    In vitro models

    Reported

    mechanism of

    action/Level

    Dose Ref

    No.

    1 Nut milk

    extract : E

    Nut Milk Extract in

    Aflatoxin B1 -induced

    Hepatocellular

    Carcinoma in

    Rats.Immunomodulat

    ory activity was

    assessed by measuring

    serum

    immunoglobulin (Ig)

    levels in control and

    experimental animals.

    Reduced IgG and

    elevated IgA and IgM

    in the

    hepatocellular

    carcinoma condition

    were returned to near

    normal levels in rats

    treated

    Adult Male

    albino wistar

    rats

    Returned to

    near normal

    levels of

    Reduced IgG

    and elevated

    IgA and IgM

    200

    mg/kg

    48

    2 Nut

    ethanolic

    extract

    (endotoxin

    free):E

    Immunomodulatory

    activity of the nut

    extracts at 1g/mL in

    mononuclear cells of

    production normal.

    Humans

    cells(Vitro)

    -Cell culture

    -Cytokine ELISA

    -Nitric oxide

    (NO) estimation

    -RT-PCR

    -Electrophoretic

    Mobility Shift

    Assay (EMSA)

    Inhibition of

    proinflammato

    ry cytokine

    production

    1g/m

    L

    49

    3 Nut milk

    extract : E

    The components of

    immune system levels

    of reactive oxygen

    species (ROS), namely

    Hydroxy radical,

    Superoxide radical,

    and H2O2 were also

    measured in spleen,

    thymus,

    and lymphocytes

    Adult Male

    albino wistar

    rats

    A significant

    increase in the

    level of LPO,

    ROS

    150

    mg/kg

    50

    4 Kalpaamr

    uthaa :F

    Kalpaamruthaa (KA)

    is a modified Siddha

    Female albino

    SpragueImmunoglobul

    ins, and

    200

    mg/kg

    51

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 25

    7.1. Balchandran Premalatha et al (1998) has reported Immunomodulatory Activity of

    Semecarpus anacardium Linn. Nut Milk Extract in Aflatoxin B -induced Hepatocellular.

    Administration of 200 mg kg- S. anacardium nut milk extract brought immunoglobulin levels

    back to near normal levels in HCC-induced rats.This indi cates the immunomodulatory effect

    of the nut milk extract in a growing tumour, and it wouldbe effective in inhibiting the growth

    of an established tumour.No significant change in immuno-globulin levels in drug control

    animals suggests that the drug did not produce any immunotoxic effects. The combined

    antitumour potency and immuno-modulatory activity of S. anacardium nut milk extract make

    it potentially useful in the treatment orprevention of immune-based diseases such as cancers.

    7.2. Singh, Divya, et al.(2006) Reported Immunomodulatory activity of Semecarpus

    anacardium extract in mononuclear cells of normal individuals and rheumatoid arthritis

    patients

    The suppressive activity of SA extract demonstrable in normal mononuclear cells was also seen

    where peripheral blood and synovial mononuclear cells of RA patients were used. Rheumatoid

    arthritis is marked by chronic synovitis and abundance of proinammatory cytokines, TNF-

    alpha IL-1beta, IL-6 and IL-12p40, produced primarily by stimulated monocytes, macrophages

    and synovial lining cells

    preparation, which has

    been formulated. It

    contains Semecarpus

    anacardium Linn.

    (SA), Emblica

    officinalis (EO), and

    honey. Synergetic

    Immunomodulatory

    activity

    Immunomodulatory

    effect of

    Kalpaamruthaa on

    7,12-dimethyl

    benz(a)anthracene-

    induced mammary

    carcinoma studied in

    rats

    Dawley rats of

    Wistar stain

    glycoprotein

    components to

    near normal

    levels

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 26

    The suppression of IL-1_ and IL-12p40 along with NF-_B and AP-1, therefore, suggests that

    SA may have important constituents that will have benets for this disease.We have also shown

    the SA suppressed LPS activated nitric oxide production by the extract in mouse macrophage-

    like cell,RAW264.7. This is not surprising since nitric oxide production is regulated by iNOS

    gene that has promoter regions for NF-KB.NO also plays a role in the pathogenesis of RA and

    therefore, its suppression by SA extract adds an additional advantage for its anti-disease

    activity

    7.3. Ramprasath, et al.(2006) Reported "Evaluation of antioxidant effect of Semecarpus

    anacardium Linn. nut extract on the components of immune system in adjuvant arthritis.

    Semecarpus anacardium Linn. Nut extract was investigated by studying the extent of lipid

    peroxidation and the activities of SAD, CAT, GPx, GSH, and ROS in the lymphocytes and

    lymphoid organs (spleen, thymus) in control and experimental rats. Anti-arthriticeffect was

    studied from the changes in the paw thickness as a measure of paw edema and arthritic score

    in arthritic and drug treated rats. In arthritic rats the extent of LPO and ROS were elevated

    profoundly and the antioxidants (SOD, CAT, GPx, GSH) were found to be significantly

    decreased. CRP levels and ESR were also found to be significantly increased in arthritic rats.

    These changes were brought back to near normal levels on treatment with the drug. No

    significant changes were observed in drug alone treated control rats. The paw thickness and

    arthritic scores were very much increased in arthritic rats, which was significantly reduced on

    treatment with the drug. These effects can be attributed to the presence of flavonoids and other

    synergistic components in the drug.

    7.4. Dharmendra et al.(2014) Immunomodulatory effect of Kalpaamruthaa on 7, 12-

    dimethyl benz (a) anthracene-induced mammary carcinoma studied in rats."

    Kalpaamruthaa (KA) is a modified Siddha preparation, which has been formulated in our

    laboratory. It contains Semecarpus anacardium Linn. (SA), Emblica officinalis (EO), and

    honey in a definite ratio. The component study of this herbal preparation revealed the presence

    of flavonoids, ascorbic acid, polyphenols, tannins, sugars, sterols, etc. Dose-dependent study

    of Kalpaamruthaa was carried out in mammary carcinoma-bearing rats, which helped us fix

    300 mg/kg body weight as the effective

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 27

    This reduction in the levels of glycoprotein components indicates that the drug KA has

    the ability to suppress malignancy by modulating the cell transformation, decreasing the degree

    of metastasis, inhibiting the progression of growth, and controlling the cancer cell proliferation

    and differentiation by causing effective favorable changes in the structure of cell membranes.

    This could be due to the cytostabilizing property of the drug and also due to inhibitory action

    of flavonoids against carcinogenesis

    In this study suggest that DMBA-induced mammary carcinoma is associated with

    immune suppression and the drug KA by means of its immunomodulating effect can serve as

    a better anticancer agent. The results also suggest that KA is found to be more effective than

    SA, which may be due to the amalgamated and additive effects of SA, E.officinalis, and honey

    present in the drug.

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 28

    8. Toxicological studies of Semecarpus anacardium.

    8.1.1.Patwarddhan et al ., 1988 have reported the toxicity of Semecarpus anacardium.

    extract

    Toxicity by oral route administration of SA with peanut oil was compared against the same

    extract emulsified with Tween-80 saline. The traditional way of administration with peanut oil

    was found to be safe and upto 25 mg/kg/day x9 days, increase in weight, RBCs & haemoglobin

    % was observed without mortality. Same dose with Tween-80 saline was found to have adverse

    effects regarding all the parameters with 16.5% mortality. This study support Ayurvedic

    method of administration for efficacy without toxicity.

    The toxicity of bibba can be controlled by administration with peanut oil or similar

    vehicles. Upto 25 mg/kg/day dose bibba can be given safely for therapeutic uses. It can act as

    a good hematinic agent and as a general tonic. Ayurvedic method of administration has shown

    reduction in toxicity with maintained efficacy.

    8.1.2. Vijayalakshmi et al.,2000 have been reported Toxic studies on biochemical

    parameters carried out in rats with Serankottai nei, a siddha drug-milk extract of

    Semecarpus anacardium nut.

    A toxicological study was carried out in rats with a Siddha preparation, milk extracts

    of SA. The effect of acute (72 h) and subacute (30 days) treatment of the drug with different

    dosage on liver and kidney functions and haematological parameters were studied. The acute

    toxicity studies with this drug did not produce mortality at any dose level given (75-2000 mg/kg

    body weight). No marked adverse alterations were observed in haematological and biochemical

    parameters during the subacute toxicity studies (50, 100, 250 and 500 mg/kg body weight). In

    the subacute treatment, the highest dose (500 mg/kg body weight) alone showed a moderate

    increase in the level of blood glucose, plasma urea, uric acid, and creatinine. In addition,

    alteration in lipid profiles were observed which may be attributed to the ghee preparation of

    the drug. Decrease in urinary urea, uric acid and creatinine levels were also observed. Histo-

    pathological examination of vital organs showed normal architecture suggesting no

    morphological disturbances.The present study shows that the Siddha preparations of S.

    anacardium nuts do not induce any toxic manipulation on the biochemical parameters

    investigated. From these one can infer and hypothesize that this drug is nontoxic and can be

    used as therapeutic agent in treating the reported diseases effectively.

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 29

    9. Conclusion

    SA reported to be a potent immunomodulator. Scientific studies have proven the efficacious role

    in preventive medicine and in the management of chronic degenerative diseases. It would

    provide an alternative to conventional chemotherapy for a variety of immunological disorder.

    Majorly the evaluation of SA as immunomodulators done by using various models and

    techniques such as; Adjuvant arthritis in rats (AIA), Lymphocyte proliferation assay,

    Measurement of induction of NO in activated macrophages, Cell culture, Cytokine ELISA.

    Recent studies found out that the SANE can inhibit proinammatory cytokine production. The

    nut milk extract is effective in restoring the fragility of lysosomal membranes in aflatoxin-

    induced hepatocellular carcinoma. The crude extract also suppressed LPS induced nuclear

    translocation. The extract also suppressed LPS activated nitric oxide production in mouse

    macrophage cell line. And the maximum tolerated dose 250 mg/kg body weight in 50%

    alcoholic extract of the fruit when given interaperitoneally to mice was found to be safe.

    Ayurvedic method of administration has shown reduction in toxicity with maintained efficacy.

    It also shows a drug interaction with propranolol against isoproterenol induced myocardial

    damage in rats.

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 30

    10. Future strategies.

    Besides mention in classic Ayurveda texts and claims of traditional physicians, it is noted that

    evidence through scientific studies is emerging to demonstrate the benefits of SA

    immunomodulation. However, the definite lacuna in all research publications reviewed in this

    report is the inadequate probing into Ayurvedic science. This has led to superficial correlations,

    where the plant drugs have remained as mere candidates for testing against selected

    pharmacological reactions. Deciphering deeper into Ayurvedic concept behind

    Immunomodulation can help identify better candidates and models for study.

    And it will help for selection of clinical and experimental studies. The later are not adequate at

    present, but it is worthwhile to project such evidences to provide lead for further studies. Drug

    discovery strategies based on natural products and traditional medicines are remerging as

    attractive options. A reverse pharmacology approach, inspired by traditional medicine and

    Ayurveda, can offer a smart strategy for new drug candidates to facilitate discovery process

    and also for the development of rational synergistic botanical formulations.

    With respect to the possible future clinical potential of immunomodulation activity is the

    observation that therapeutic dosing with SA inhibited established arthritis in rats. The plant-

    derived immunomodulators thus have tremendous future potential for developing new

    pharmaceutical products. Phytochemical analysis of these SANEs is in progress to identify

    bioactive molecules responsible for immunomodulatory properties in these plants.

  • Exploring therapeutic Potential of Bhallataka for Immunomodulatory activity | 31

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