Lipid lowering activity of a polyherbal extract in

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Submitted to



In partial fulfillment

of the requirement for the Degree of

Master of Pharmacy in Pharmacology


Under the Guidance of


Professor & Head

Department Of Pharmacology








Name of the Candidate

and address

Patibandla Suraj,, Bathula enclave, Gandhipuram-3, Rajahmundry,

Andhra Pradesh-533103.


Name of the Institution

Dayananda Sagar College of Pharmacy,

Kumaraswamy Layout,

Bangalore – 560 078,

Karnataka, India.


Course of Study and


M. Pharmacy-Pharmacology


Date of Admission

26 August 2012



Brief resume of the intended work:

6.1 – Need of the study:

Cardiovascular diseases (CVDs) have significantly contributed to the number of global deaths, whereas, by the year 2020, it is expected that CVDs will become the leading cause of death and disability worldwide. At normal levels the lipids have been noted to perform important functions in the body, but may cause various health problems if present in excess amounts. The term hyperlipidemia refers to the elevated lipid levels in the body including high cholesterol and high triglyceride levels. However, the cholesterol circulates in the blood stream and is involved in the structure and function of the cell, whereas, the triglycerides are either used immediately or stored in the fat cells. Further, High cholesterol levels in the body have been considered as a modifiable risk factor which is evident by fact that plasma cholesterol at levels greater than 200mg/dl causes 4.4 million deaths in a year. Various types of cholesterol have been reported that include total cholesterol (TC), consisting of all the cholesterols combined; HDL cholesterol, often referred to as good cholesterol; and LDL cholesterol, often called bad cholesterol. The abnormal cholesterol levels are a result of unhealthy lifestyle including taking high-fat diet and other lifestyle factors like being overweight, heavy alcohol use and lack of exercise. Moreover, diabetes and underactive thyroid gland have also been reported to cause high cholesterol levels. Other illnesses that may elevate cholesterol levels include polycystic ovary syndrome and kidney diseases. The higher levels of female hormones like oestrogen, have been noted to increase or change cholesterol levels. Number of studies have shown statins, niacin analogues, fibrates, bile acid binding resins and cholesterol absorption inhibitors to possess certain modulatory roles in the treatment of hyperlipidemia.1

Hyperlipidemia is characterized by elevated serum total cholesterol, low density lipoprotein, very low density lipoprotein and decreased high density lipoprotein levels. Hyperlipidemia associated lipid disorders are considered to cause atherosclerotic cardiovascular disease. Currently available drugs have been associated with number of side effects. The consumption of synthetic drugs leads to hyperuricemia, diarrhoea, nausea, myositis, gastric irritation, flushing, dry skin and abnormal liver function.2


Plant Profile :

Dolichos biflorus Linn., Syn. Dolichos uniflorus (Family- Fabaceae) is a branched, sub-erect and downing herb, native to most parts of India and is found up to altitudes of 1000 m. It is a fast growing annual vine with trifoliate leaves and brown, flat, curved pods filled with seeds. The seeds can be cooked and eaten. In Ayurveda, the seed is used in the treatment of piles, pain, constipation, wounds, urinary calculi, cough, edema, asthma etc. The soup prepared from seeds is also beneficial in enlarged liver and spleen. The seeds of D. biflorus have been reported to show antilithiatic, antihepatotoxic and hypolipidemic activity and involved in lowering the level of blood sugar and total cholesterol.3
Fenugreek (Trigonella foenum graecum Linn.) has been used as a cooking spice and flavoring agent for centuries and it is a member of Leguminosae family. In Ayurveda and Siddha, Methi has been mentioned as Tikta ras, Ushanveeryam, Vata-Kalphaharam, in fevers, dysentery. In Unani, it is described as resolvant, aphrodiasic, diuretic, emenagogue, expectorant in bronchitis, piles, externally in inflammatory

conditions. Hypolipidemic activity of this herb may be attributed due to presence of steroidal saponins (diosgenin, yamogenin, tigogenin and neotigogenin), alkaloids (mainly trigonelline) and free amino acids.4

M. koenigii (L.) Spreng commonly known as ‘Curry Patta’ is a tropical to sub-tropical small tree in the family Rutaceae which is native to India. It is traditionally used in India as a spice for its characteristic flavor and aroma. The aromatic leaves are considered as a tonic, anthelmintic, analgesic, digestive, and appetizer, being widely used in Indian cookery for flavoring food stuffs. The leaves have been used for the treatment of piles, inflammation, itching, fresh cuts, dysentery, vomiting, burses and dropsy. The aqueous extracts of M. koenigii have a strong hypolipidemic activity and have been indicated for the treatment of the mild form of diabetes. M. koenigii leaves contains a range of active pharmacological agents, including carbazole alkaloids, flavonoids, furanocoumarins, terpenoids and tannins. Leaf extracts of M. koenigii and 4 carbazole alkaloids isolated from them show Antihyperlipidemic activity.5

6.2 – Review of the literature:

  1. Wound healing activity of Murraya koenigii leaf extract using excision wound model has been reported.6

  1. Immunomodulatory activity of methanolic extract of Murraya koenigii has been reported to be evaluated on humoral and cell mediated immune response to ovalbumin, phagocytic activity by carbon clearance test.7

  1. Anti-inflamatory activity of Murraya koenigii leaves using carrageenan induced paw oedema method has been reported.8

  1. Anti-helminthic activity of methanolic extract Murraya koenigii against Indian earth worms has been reported.9

  1. Anti-bacterial and cytotoxic activities of leaf extract of Murraya koenigii has been evaluated by disk diffusion technique and brine shrimp lethality bioassay respectively.10

  1. Hepatoprotective activity of extract of Murraya koenigii bark against carbon-tetrachloride induced hepatotoxicity has been reported.11

  1. Analgesic activity of Murraya koenigii leaf extract in rats by hot plate method has been evaluated.12

  1. Anti-oxidant and antiradical activity of Dolichos biflorus seed extract has been evaluated.3

  1. Antilithiatic activity of seeds of Dolichos biflorus and rhizomes of Bergenia lingulata In vitro has been evaluated.13

  1. Antihepatotoxic activity of kulthi (Dolichos biflorus) seeds in paracetmol intoxicated rats has been evaluated.14

  1. Chemomodulatory effect of Dolichos biflorus on skin and fore stomach papillomagenesis in swiss albino rats has been reported.15

  1. Analgesic and anti-inflammatory activities of Trigonella foenum seed extract has been reported.16

  1. Estrogenic activity of Trigonella foenum seed extract has been reported.17

  1. Anti-diabetic activity of Trigonella foenum seeds leaves has been evaluated clinically.18

  1. Anti-oxidant activity of Trigonella foenum using various In vitro and Ex vivo models has been evaluated.19

16. Anxiolytic activity of Trigonella foenum seed extract is due to the bioactive principles which possess anxiolytic activity.20

  1. Active compounds present in the Trigonella foenum seed extracts possessing CNS stimulant and depressant activity has been reported.21

  1. Fungicidal activity of Trigonella foenum seed extract on Malassezia globosa has been evaluated.22

  1. Antineoplastic Effect of Trigonella foenum seed extract against Acute Myeloblastic Leukemia Cell line has been evaluated.23

  1. Adaptogenic activity of Trigonella foenum seed extract in rodents exposed to anoxia and immobilization stress has been reported.24

6.3 – Objective of the study:

The objective of the present investigation is to evaluate the Antihyperlipidemic activity of polyherbal extract against acute and chronic hyperlipidemia.

The work consists of :-

  1. Collection and authentication of the plant.

  2. Extraction of required plant part using soxhlet apparatus.

  3. Preliminary Phytochemical Screening and acute toxicity studies of the extracts.

  4. Evaluation of lipid lowering effect of polyherbal extract using in vivo methods.

Materials and methods:

7.1 Source of data:
Journal of Ethnopharmacology, Indian journal of forestry, International journal of pharmacology, Indian journal of experimental biology, Indian journal of physiology and pharmacology.

Web site :

Standard books:

Indian Materia Medica

Experimental pharmacology by M.N.Ghosh

Medicinal plants research

7.2 Methodology :

Method of collection of the data:

The Experiments will be conducted using different animal models and the data will be generated from such experimental studies.

Collection and authentification of Polyherbal plant parts:

The seeds, leaves will be collected and authenticated by botanist.

Extraction :

The freshly aquired plant part of the three herbs will be dried at room temperature. Dry material will be coarsely pulverized to powder form seperately. Each of the dried powders will be subjected to extraction with methanol using soxhlet extractor. The methanolic extract will be concentrated below 40oC using rotary flash evaporator.7

The three extracts will be combined in equal proportions and administered in the animals, as a suspension in 1% CMC for the evaluation of anti-hyperlipidemic activity.


I) Acute toxicity studies will be carried out as per OECD guidelines. The therapeutic safe lower and higher dose of the extract will be fixed based on the result of the acute toxicity studies.

II) Evaluation of antihyperlipidemic activity in wistar strain albino rats will be done using following methods:

In the experiment a total no. of 48 rats will be used for each model. The animals will be divided into 8 groups of six each.

  1. Acute study

Triton WR-1339 induced hyperlipidemia in rats:

Group1 (G1) : Normal control

Group2 (G2) : Hyperlipidemic control

Group3 (G3) : Polyherbal extract (Lower dose)

Group4 (G4) : Polyherbal extract (higher dose)

Group5 (G5) : D. biflorus seed extract

Group6 (G6) : M. koenigii leaf extract

Group7 (G7) : Trigonella foenum seed extract

Group8 (G8) : Standard (10mg/kg) Atorvastatin

All animals except normal control group (G1) will be injected with Triton at a dose of 400 mg/kg to achieve hyperlipidemia.

  1. Chronic study-

Cholesterol- diet induced hyperlipidemia in rats:

Group1 (G1) : Normal control

Group2 (G2) : Hyperlipidemic control

Group3 (G3) : Polyherbal extract (Lower dose)

Group4 (G4) : Polyherbal extract (higher dose)

Group5 (G5) : D. biflorus seed extract

Group6 (G6) : M. koenigii leaf extract

Group7 (G7) : Trigonella foenum seed extract

Group8 (G8) : Standard (10mg/kg) Atorvastatin
All animals except normal control group (G1) will be fed with cholesterol rich diet for 28 days to achieve hyperlipidemia.

Using these models, the lipid lowering (Hypolipidemic) effect of the extracts will be investigated.25

Parameters to be evaluated:

In both acute and chronic hyperlipidemia studies, after the experimental period, all animals will be sacrificed and analysed for the biochemical parameters in serum and liver tissue and also for histopathological changes which will be carried out by standard procedure.

  1. Biochemical analysis ( Serum & Liver tissue):

  1. Total cholesterol

  2. Triglycerides

  3. Phospholipids

  4. HDL cholesterol levels

  5. LDL cholesterol levels

  6. VLDL cholesterol levels

  1. Histopathological studies

Expected outcome: From the above observations, a conclusion may be drawn on the potential of the polyherbal extract on experimental hyperlipidemia in animal models which can be extrapolated to human beings.

Statistical analysis: The results will be expressed as Mean ±SEM. The data will be evaluated using one way ANOVA followed by Newmann-keulus multiple range test & differences below p<0.05 will be considered as significant

7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If so, Please describe briefly.

Yes, the study requires investigation on Wistar strain albino rats.

7.4 Has ethical clearance been obtained from your institution in case of 7.3?

Yes, the ethical clearance certificate is attached.



  1. Ankur Rohilla, Nidhi Dagar, Seema Rohilla, Amerjeet Dahiya, Ashok Kushnoor. Hyperlipidemia- a deadly pathological condition. Int J Curr Pharm Res. 2012; 4:15-18.

  2. SS Sudha, R Karthic, Naveen, J Rengaramanujan. Anti Hyperlipidemic activity of Spirulina platensis in Triton x-100 induced hyperlipidemic rats. Hygeia J D med. 2011; 3(2):32-37.

  3. Bibhabasu Hazra, Rhitajit Sarkar, Sourav Mandal, Santanu Biswas and Nripendranath Mandal. Studies on antioxidant and antiradical activities of Dolichos biflorus seed extract. Afr J Biotechnol. 2009; 8(16):3927-3933.

  4. Amish J Patel, Natvarlal M Patel, Amit A Patel, Jitendra Patel. Interaction of Trigonella foenum-graceum and Acacia catechu for Antihyperlipidemic activity. JPSBR. 2011; 1:54-58.

  5. Rahul Birari, Vishal Javia, Kamlesh Kumar Bhutani. Antiobesity and Lipid lowering effects of Murraya koenigii Spreng leaves extracts and mahanimbine on high fat diet induced rats. Fitoterapia. 2010; 81(8):1129-1133.

  6. Dinesh kumar Patidar, Narendra Yadav, Vinod Nakra, Pradeep Sharma, Ajay Bagherwal. Wound healing activity of Murraya koenigii leaf extract. Pharmacie Globale {ijcp}. 2010; 4(09):1-2.

  7. Abhishek S Shah, Alok S Wakade & Arachana R Juvekar. Immunomodulatory activity of methanolic extract of Murraya koenigii Spreng. Leaves. Indian J Exp Biol. 2008; 46:505-509.

  8. Vaibhav M Darvekar, Vijay R Patil, Amol B Choudhari. Anti-inflammatory activity of Murraya koenigii Sperng on experimental animals. J Nat Prod Plant Resour. 2011; 1(1):65-69.

  9. Anil Kumar, Ashok Thripathi, Jyotsana Dora, Rishikanth Thripathi. Anti-helmenthic activity of methanolic extract of Murraya koenigii leaves (Linn). Int J res Pharm & Biomed Sci. 2011; 2(4):1698-1700.

  10. Biswa Nath Das, Bishyajit Kumar Biswas. Antibacterial and cytotoxic activities of the leaf extract of Murraya koenigii. Int J Life Sc Bt & Pharm Res. 2012, 1(3):59-63.

  11. MS Pande, SPBN Guptha, A Pathak. Hepatoprotective activity of Murraya koenigii Linn bark. J Herb Med Tox. 2009; 3(1):69-71.

  12. WD Ratnasooriya, LDC Peiris, AS Amerasekera. Analgesic activity of Murraya Koenigii leaf extract in rats. Med Sci Res. 1994; 22:837-840.

  13. Garimella TS, Jolly CI, Narayan S. In vitro studies on antilithiatic activity of seeds of Dolichos biflorus Linn. and rhizomes of Bergenia ligulata Wall. Phytother Res. 2001; 15(4):351-5.

  14. Laskar S, Bhattacharya UK, Sinhababu A, Basak BK. Antihepatotoxic activity of Kulthi (Dolichos biflorus) seed in rats. Fitoterapia. 1988; 69:401-402.

  15. Nanta R, Kale RK. Chemomodulatory effect of Dolichos biflorus linn. on skin and forestomach papillomagenesis in Swiss albino mice. Indian J Exp Biol. 2011; 49(7):483-90.

  16. Vyas S, Agarwal RP, Solanki P, Trivedi P. Analgesic activity and anti inflammatory activities of Trigonella foenum graecum (seed) extract. Acta Pol Pharm. 2008; 65(4):473-6.

  17. S Sreeja, VS Anju, S Sreeja. In vitro esterogenic activities of fenugreek Trigonella foenum graecum seeds. Indian J Med Res. 2010; 131:814-819.

  18. Mohammad Yaheya Mohammad Ismail. Clinical evaluation of antidiabetic activity of Trigonella seeds and Aegle marmelos leaves. World Appl Sci J 2009; 7(10):1231-1234.

  1. N Subhashini, A Thangathirupathi, N Lavanya. Antioxidant activity of Trigonella foenum graecum using various In vitro and Ex vivo models. Int J Pharm Sci. 2011; 3(2):96-102.

  2. M Iyer, H Belapurkar, O Sherikar, SB Kasture. Anxiolytic activity of Trigonella foenum graecum seeds. J Nat Rem. 2004; 4(1):61-65.

  3. B Natarajan, A Muralidharan, R Satish, R Dhananjayan. Neuropharmacological activity of Trigonella foenum graecum Linn.seed. J Nat Rem. 2007; 7(1):160-165.

  4. Shubhangi Mugali, Renu Mishra, Rajana, Shikha Mandloi. Fungicidal activity of Trigonella foenum graecum seeds on Malazssezia globosa. Nanobiotecnia Universale. 2011; 2(1):27-32.

  5. Alizadeh et al. Antineoplastic effect of Fenugreek (Trigonella foenum graecum) seed extract against Acute Myeloblastic Leukemia cell line (KG-1).Iran J Blood Cancer. 2009; 4:139-146.

  6. Vinod S Pawar, Shivakumar Hugar. Adaptogenic activity of Trigonella foenum graecum (Linn) seeds in rodents exposed to anoxia and immobilization stress. Asian Pac J Trop Biomed. 2012,S208-S211.

  7. Parasuram S, Kumar EP, Anil Kumar, Emerson SF. Anti-hyperlipidemic effect of Triglize, a polyherbal formulation. Int J Pharmacy Pharm Sci. 2010; 2:118-122.


Signature of the candidate

(Patibandla Suraj)


Remarks of the Guide:

Requested for clearance and approval


Name and Designation of

11.1 Guide:
11.2 Signature:

Dr. Geetha K M

Professor & Head

Dept of Pharmacology

Dayananda SagarCollege of Pharmacy,

Kumaraswamy layout, Bangalore – 560 078.

11.3 Co-Guide:

11.4 Signature

Not applicable

11.5 Head of the Department:
11.6 Signature

Dr. Geetha K.M.

Professor & Head

Dept of Pharmacology

Dayananda Sagar College of Pharmacy,

Kumaraswamy layout, Bangalore – 560 078.


12.1 Remarks of the Chairman and


Recommended for research

12.2 Signature

Dr. V. Murugan

Professor and Principal

Dept of Pharmaceutical Chemistry

Dayananda SagarCollege of Pharmacy,

Kumaraswamy layout, Bangalore – 560 078.

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