Anticancer effect of methanol extract of Hippophae rhamnoides Linn seed alone and in combination with Cyclophosphamide




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Anticancer effect of methanol extract of Hippophae rhamnoides Linn. seed alone and in combination with Cyclophosphamide”

M. Pharm Dissertation Protocol Submitted to



Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore– 560 041
By

Mr. Chirag A. Patel B.Pharm

Under the Guidance of
Dr. Divakar Goli M.Pharm. Ph.D

Professor







2008-09

Department of Pharmacology,

Acharya & B.M. Reddy College of Pharmacy,

Soldevanahalli, Chikkabanavara (Post)

Hesaraghatta Main Road, Bangalore – 560 090

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE
ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION




1.

Name of the candidate &

Address.



Mr. Chirag A. Patel

At & Post-Aritha,

Ta-Sami, Dist-Patan, Patelvas,

Gujarat-384240




2.


Name of the Institution.



Acharya & B.M. Reddy College Of Pharmacy

Soldevanahalli, Hesaraghatta Road,

Chikkabanavara Post,

Bangalore-560090.

Phone No: 080 65650815

Fax No: 080 28393541



3.

Course of the study

& subject

M.Pharm (Pharmacology)





4.

Date of admission.


05/06/2008




5.

Title of the Topic



Anticancer effect of methanol extract of Hippophae rhamnoides Linn. seed alone and in combination with Cyclophosphamide”


6.

Brief resume of intended work




  1. Introduction and need of the work




  1. Review of Literature




  1. Aim and Objective of the study

Enclosure I


Enclosure II
Enclosure III



7.

Materials & Methods




  1. Source of data




  1. Methods of collection of data




  1. Does the study require investigation on animals?

    1. If yes give details




  1. Has ethical clearance been obtained from your institution in case of 7.3

Enclosure IV


Enclosure V
Enclosure VI

Yes (Copy Enclosed)




8.

List of references ( About 1 – 6)


Enclosure VII





9.

Signature of the candidate








10.

Remarks & Signature of the guide






11.

Name & Designation of Guide





Dr. Divakar Goli M.Pharm. Ph.D

Professor

Acharya & B.M. Reddy College of Pharmacy,

Soldevanahalli, Chikkabanavara (Post)

Hesaraghatta Main Road Bangalore – 560 090


12.

Head of the Department

Signature of HOD



Dr. Kalyani Divakar M.Pharm. Ph.D

Professor & Head Dept of Pharmacology,

Acharya & B.M. Reddy College of Pharmacy,

Soldevanahalli, Chikkabanavara (Post)

Hesaraghatta Main Road Bangalore– 560 090



13.

Remarks of the Principal






14.

Signature of Principal




Dr. Divakar Goli M.Pharm. Ph.D

Principal

Department of Pharmacology,

Acharya & B.M. Reddy College of Pharmacy,

Soldevanahalli, Chikkabanavara (Post)

Hesaraghatta Main Road Bangalore – 560 090


Enclosure-I

6. BRIEF RESUME OF INTENDED WORK

6.1 Introduction and need of work:

Neoplasia literally means “new growth”. A neoplasm as defined by Willis is “an abnormal mass of tissues, the growth of which as same of the normal tissue and persist in the same excessive manner after the cessation of the stimuli which evoked the change”.1

In common medical usage, neoplasm is often referred to as tumor, and the study of tumors is called oncology. Characteristic of benign & malignant neoplasm include 1) Differentiation & Anaplasia 2) Rate of growth 3) Local invasion and 4) Metastasis. Malignant tumors are collectively referred to as cancer. Cancer is not one disease but many disorders that share profound growth deregulation, some cancers such as Hodgkin’s lymphoma is curable whereas cancer of pancreas has high mortality.1

Cyclophosphamide is an alkylating chemotherapeutic agent with immunosuppressive activities. The alkylating metabolite can bind to a variety of molecules including amino acids, proteins, and peptides, but the most important binding site is DNA where cross-linking occurs.2 It is effective in the treatment of cancer (lymphoma, acute and chronic leukemia’s, multiple myeloma) and non-malignant disease states such as rheumatoid arthritis and vasculities. It is inactive until metabolized in the liver by p450 mixed function oxidase. The important side effects are nausea, vomiting and bone marrow depression.4

In fact, one of the most striking medical practices of the 21st century is the chemoprevention of cancer.5 Many plant species are known to elicit antimutagenesis and thus have a full range of prospective application in human health. Even for populations which use herbs traditionally, encouraging the use of species with chemopreventive actions could be helpful as part of life expectancy improvement strategies: costs are significantly low, herbs have usually little or no toxicity during long-term oral administration and are relatively available at large scale.6

Seabuckthorn (Hippophae rhamnoides L. Elaegnaceae) is thorny nitrogen fixing deciduous shrub, native to Europe and Asia. All parts of the plant are considered to be a good source of a large number of bioactive substances. The ripe fruit has been reported to be a rich source of Vitamins A, C, E, and K, carotenoids, and organic acids. Many medicinal effects of Seabuckthorn against antihypertensive, inflammatory disease, cardiovascular diseases, mucosal injuries, and skin disorders have been suggested to be due to the high contents of antioxidant substances present in this plant.7

The present study is planned to evaluate anticancer activity of methanol extract of Seabuckthorn seeds alone and in combination with Cyclophosphamide.

Enclosure-II

6.2 Review of Literature:

Plant:

Name of the plant selected for the present study isHippophae rhamnoides L”, belonging to family Elaeagnaceae.



Description of plant:

Seabuckthorn is a dioecious multi-branched, thorny shrub, reaching 2 to 4 m in height with stout branches forming a round often symmetrical head. It has brown or black rough bark and a thick grayish-green crown. Staminate and pollinate flowers are inconspicuous appearing before the leaves. Leaves are alternate, narrow 4 to 6 cm long, and lanceolate with a silver-grey color on the upper side. Flower buds are formed mostly on 2-year-old wood, differentiated during the previous growing season. Fruit is subglobose, 6 to 10 mm long and 4 to 6 mm in diameter, turning yellow to orange when mature in mid September. The root system is characterized by nitrogen fixing nodules. The male inflorescence consists of four to six apetalous flowers. The female inflorescence usually consists of one single apetalous flower with one ovary and one ovule.8-9



Distribution of plant:

Seabuckthorn (Hippophae rhamnoides) is one of the important natural resources of the mountainous regions of China and Russia. The plant grows naturally in sandy soil at an altitude of 1,200-4,500 meters (4,000-14,000 feet) in cold climates, though it can be cultivated at lower altitudes and into temperate zones. Seabuckthorn is a widely distributed throughout the temperate zone of Asia and Europe. The distribution ranges from Himalayan regions including India, Nepal, Bhutan, Pakistan and Afghanistan to China, Mongolia, Russia, Kazakhstan, Hungary, Romania, Switzerland, Germany, France and Britain. The wide distribution of Seabuckthorn is reflected in its habit-related variation not only in morphology, yield, growth rhythms and cold hardiness, but also in berry related characters such as fresh weight, chemical and sensory attributes.10



Chemical Constituents:

Seabuckthorn berries are among the most nutritious and vitamin-rich fruits found in the plant kingdom. The vitamin C concentration in berries varies from 360 mg/100g of berries and Vitamin E concentration can be up to 160 mg/100g of berries. Seabuckthorn is also rich in flavonoid and contains appreciable amounts of water soluble and fat soluble vitamins. Seabuckthorn berries contain up to 13% soluble sugars, mainly glucose, fructose, xylose and 3.9% organic acids, mainly malic and succinic acid. A total of 18 amino acids have been found in Seabuckthorn fruit. There are at least 24 chemical elements present in Seabuckthorn juice; e.g. Nitrogen, Phosphorous, Iron, Manganese, Boron, Calcium, Aluminum, Silicon and others. Oil from the juice and pulp is rich in palmitic and palmitoleic acids (C16:0 and C16:1), while the oil from the seed contains unsaturated fatty acids of C18 type oils (linoleic and linolenic acid). Oils from the seed and juice also contain Vitamin E and carotenes. In addition, sea-buckthorn berries, leaves and bark contain sitosterol, tocopherol and many other bioactive compounds.10


Traditional Uses:


Different parts of H. rhamnoides L. have been used for the treatment of diseases in traditional medicine in various countries of the world. It has long been used for relieving cough, aiding digestion, invigorating blood circulation and alleviating pain since ancient time. The extracts of H. rhamnoides L. branches and leaves are used to treat colitis in humans and animals. Branches and leaves are also used in the treatment of diarrhea. Leaves are used in gastrointestinal and dermatologic disorders and have been applied as compress form in rheumatoid arthritis in the middle Asia flowers of H. rhamnoides L. are used as skin softener in Tajikistan. For its haemostatic and anti-inflammatory effects, fruits of the plant are added to prescriptions in pulmonary, gastrointestinal, cardiac, blood and metabolic disorders in Indian and Tibetian medicine. After reviewing the ancient literature Li and Guo point out that sea buckthorn is a mild drug with the characteristic effects such as lowering fever, diminishing inflammation, counteracting toxicity and abscesses, treating cough and colds, keeping warm, easing respiration, clearing sputum, having mildly laxative effect, treating tumors, especially of the stomach and the esophagus, and treating different kinds of gynecological diseases in Tibetian medicine. Oil extracts obtained from fruits are used in liver diseases, inflammatory processes, absorption disorders in the gastrointestinal system, and are applied externally in hemorrhage. Juice, syrup, and oil of the fruits have been used as pain killer, to heal wounds, in ulcer and other diseases of the stomach, cancer, and as a metabolism regulator in traditional medicine. The freshly pressed juice is used in the treatment of colds, febrile conditions, and exhaustion. Oil from fruits and seeds is used in the treatment of eczema, lupus erythematosus, chronic wounds that are difficult to heal, inflammatory diseases, erosion of the cervix uteri, in the treatment of burns and frozen parts of the body. Also, its berry oil is reported to treat skin disease and thrombosis. Oil extracts are used externally in dermatologic diseases such as eczema, psoriasis, lupus erythematosus, and chronic dermatoses. In ophthalmology, they are used in the treatment of keratitis, trachoma, injuries or burns of eye lid, conjunctivitis.11

Therapeutic uses with scientific support:

  • Antioxidant activity was performed using alcoholic extracts of leaves, fruits and berries of Seabuckthorn (SBT) against chromium (VI), sodium nitroprusside, and hypoxia induced oxidative stress in albino rats. The results showed that the leaves, fruits and berries of Seabuckthorn (SBT) have marked anti-oxidant activity.12-14

  • Antioxidant and antibacterial activities were conducted using methanol extracts of various Seabuckthorn (H. rhamnoides L.) seeds. The results indicated that antioxidant and antimicrobial effects of the extract implicate its potential for natural preservation.15-16

  • Anti-platelet aggregation activity was conducted using supercritical CO2 extracted Seabuckthorn berry oil (SBO) and Total flavones of Hippophae Rhamnoides L (TFH). The study reveled that the beneficial effects of SBO on blood Clotting.17-18

  • Anti-mutagenic activity was conducted using methanol extract of Seabuckthorn (H. rhamnoides Linn) berries with Ames assay. The results reveled that Seabuckthorn berries has Anti-mutagenic activity.19

  • In vitro anti-tumor activity conducted using isorhamnetin flavonol aglycone isolated from Hippophae rhamnoides L. against BEL-7402 cells. The treatment results indicated that in the appearance of a hypodiploid peak (sub-G0/G1 peak), probably due to the presence of cells in apoptosis and apoptotic bodies with DNA content less than 2n.20

  • Anti-inflammatory activity was conducted using leaves and berries extract of Seabuckthorn (SBT). The observations suggest that the SBT leaves extract has a significant anti-inflammatory activity and had the potential for the inflammatory diseases.21-23

  • Anti ulcer activity conducted using CO2-extracted seed and pulp oils of Sea buckthorn (Hippophae rhamnoides L.) on experimental models of rats. The results suggested that Seabuckthorn seed and pulp oils have both preventive and curative effects against experimental gastric ulcers in rats.24

  • Seabuckthorn extract appraised the effect on liver fibrosis by significantly decreasing the serum levels of LN (Laminin), HA (Hyaluronic acid), collagen types III and IV, total bile acid (TBA). So Seabuckthorn may be a hopeful drug for prevention and treatment of liver fibrosis.25

  • Antihypertensive effect was conducted using total flavones extracted from seed residues of Hippophae rhamnoides L. (TFH-SR) and its underlying mechanism in chronic sucrose-fed rats by evaluating its ability to regulate insulin and angiotensin-II levels.26

  • Cardiac function and hemodynamic effects of total flavones of Hippophae rhamnoids L (TFH) performed on healthy human subjects. The results suggested that TFH may not only improve myocardial contractility and strengthen cardiac pump function, but also may decrease peripheral vessels resistance and increase vessels elasticity.27

  • Healing activity was performed using seed oil, total flavones, procyanidins and leaf extract of Seabuckthorn (Hippophae rhamnoides) against hypoxia induced cerebral vascular injury, stress of patellar tendon, acetic acid-induced lesions in the rat stomach and cutaneous excision-punch wound model respectively. The results suggest that Seabuckthorn promotes wound healing which may be due to increased antioxidant levels in the granulation tissue.28-31

  • Glycometabolism activity performed using flavonoid from the seed residue and fruit residue of Hippophae rhamnoides L. (FSH and FFH) in mice. The results indicated that FSH and FFH can decrease the levels of blood glucose and lipid in normal mice, and also control of glyconeogenesis.32

  • Radioprotection activity conducted using berries and herbal preparation of Hippophae rhamnoides against whole body lethal Irradiation in mice. The results indicated that the H. rhamnoides protects mitochondrial and genomic DNA from radiation-induced damage.33-35

  • Effect of dietary supplementation with Seabuckthorn (Hippophae rhamnoides) seeds and pulp oils on fatty acid composition of skin glycerol phospholipids of patients with atopic dermatitis. The results show that the fatty acid composition of skin glycerol phospholipids is well buffered against short-term dietary modification.36
  • Protective effects of seed oil and fruit extracts of Seabuckthorn (Hippophae rhamnoides L.) against injury induced by sulfur dioxide inhalation and the toxic effects of arsenic in mice respectively.37-38



Enclosure-III

6.3 Aim and Objectives of the study

Aim:

The present study is intended to investigate the anticancer effect of methanol extract of Hippophae rhamnoides Linn. seeds alone and in combination with Cyclophosphamide.


Objectives:

  1. To collect and prepare the methanol extract of seeds of Hippophae rhamnoides L.

  2. To perform preliminary phytochemical screening of methanol extracts of seeds Hippophae rhamnoides L.

  3. To study the acute toxicity study of methanol extract of seeds of Hippophae rhamnoides L.

  4. To study the anticancer effect of methanol extract of seed Hippophae rhamnoides L. alone and in combination with Cyclophosphamide through:




          1. MTT assay

          2. Clonogenic Assay

          3. Cell viability test

          4. DNA fragmentation test

          5. Glutathione estimation

          6. Ames Assay


Enclosure-IV
7. Materials and Methods

7.1 Source of Data:

Data will be obtained from laboratory based studies by finding out-



  1. Phytochemical analyses of methanol extract of seed Hippophae rhamnoides Linn.

  2. Evaluations of the anticancer effect of methanol extract of seed Hippophae rhamnoides Linn. alone and in combination with Cyclophosphamide by conducting:

    1. Glutathione estimation (in Swiss albino mice).

    2. MTT assay, Clonogenic assay and Viability test on HL-60 Cell lines

    3. DNA fragmentation test on HL-60 Cell lines

    4. Ames assay.

  3. National & International Journal

  4. Text books.

  5. Internet.


Enclosure-V

7.2 Method of Collection of Data

Drug: Cyclophosphamide will be purchase from pharmacy store.

Plant: The seed of Hippophae rhamnoides L. will be collected and dried. They will be then coarsely powdered & stored in an air tight container.

Animals: Swiss albino mice weighing 22-25 g will be selected for glutathione estimation. The animals will be housed in standard environmental condition and provided with food and water at ad libitum.

Solvent: Methanol is selected for extraction of the seed powder.

Methodology:

Acute toxicity study of methanol extract of seeds of Hippophae rhamnoides L: - Acute toxicity study will be conduct according to OECD guideline 425.

Preparation of Extraction: - Powdered seed of Hippophae rhamnoides L will be extracted by using methanol for 18 h with Soxhlet extractor. The dried extract will be used for determination of the anticancer activity.

Pharmacological studies: - Evaluations of the anticancer effect of methanol extract of seeds of Hippophae rhamnoides L. alone and in combination with Cyclophosphamide by conducting following methods:



  1. MTT assay

  2. Clonogenic Assay

  3. Cell viability test

  4. DNA fragmentation test

  5. Glutathione estimation

  6. Ames Assay


MTT Assay:-39

The 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a common method used to assess cell proliferation and cytotoxicity. Briefly, 1x104 exponentially growing cells will be exposed to various concentrations of drug and incubated and MTT will be added and incubated at 37oC for 4 h. The precipitated Formosan salt will be dissolved in DMSO and the samples will be read at 570 nm. The 50% inhibitory concentration (IC50) of drug will be calculated.



DNA fragmentation analysis:- 39

HL-60 cells will be incubated with 0, 5, 10, 15, 20 and 25 μg/ml of drug for 24 h at 37oC. The DNA will be isolated and precipitated with ethanol, air-dried and dissolved in TE buffer (5 mM Tris-HCl (pH 8.0) and 20mM eidetic acid. DNA fragmentation will be analyzed by agarose gel electrophoresis.



Cell viability:-39

The effect of drug on the HL-60 cancer Cell will be determined by means of tryphan blue exclusion technique. Briefly, 3x104 cells will be seeded per well in a 24 well and then treated with various concentrations of drug. The wells will be incubated at 37oC and the number of cultured cells in the different wells will be counted using a hemocytometer after staining with 0.4% tryphan blue every 24 h to calculate the doubling time.



Clonogenic Assay:- 40

Tumor colony forming units will be cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 0.3% agar and 20% fetal calf serum. The cultures will be incubated at 37°C in a fully humidified atmosphere containing 10% CO2 in air. Colonies (greater than 40 cells) will be scored after 10-20 days using a dissecting microscope at ×32. A linear relation of the number of cells plated and colonies could be established.


Ames assay:- 19

The salmonella point mutation assay proposed by Maron and Ames was followed with little amendments as suggested by Bala and Grover to verify the inhibitory activity of the extract.

Constant concentration of two direct acting mutagens, NPD (20 µg/0.1 ml/plate), sodium azide (2.5 g/0.1 ml/plate) and S9 dependent mutagen, 2AF (20 µg /0.1 ml/plate) will be used to positive control. 0.1 ml each of bacterial culture (about 1-2 X108 cells/ml), mutagen and extract will be added to 2 ml of top agar. Equal quantity of the mutagen and the extract will be blended and allowed to stand for 30 min at 37˚C under incessant shaking and 0.2 ml of this will be added to 2 ml of soft agar with 0.1 ml of fresh bacterial culture. Soft agar will be pour on minimal glucose agar plate at 37˚C for 48 h. concurrently, a positive control and a negative control will also be set. All the test sample and mutagen, i.e. NPD and 2AF, will be dissolved in dimethylsulfoxide and sodium azide in distilled water. The activity of each extract will be expressed as percentage decrease of reverse mutations

Glutathione estimation:- 2

The tumor will be maintained in vivo by serial intraperitoneal (i.p.) transplantation of 1 × 107 tumor cells (0.25 ml in phosphate-buffered saline, pH 7.4) per mice.



Drugs treatment schedule and antitumor activity:-

Tumor-transplanted mice will be randomly divided into four groups, (I-V) of 20 mice each. Group-I mice will be serve as control and received normal saline. Group-II and Group-III mice will be given 1/3 of LD50 and 1/7 of LD50 of methanolic extract of Seabuckthorn from 5th day to the 10th day of tumor transplantation respectively. Group-IV mice will be given a single dose of CP, i.p., on the 10th day post-tumor transplantation. Group-V mice will be given methanolic extract of Seabuckthorn and, then, administered with CP on the 10th day post-tumor transplantation. The survival patterns of the hosts will be determined and deaths, if any, in different groups, will be recorded daily. After 1, 3, 6 and 12 days of treatment, liver, kidneys, spleen, and tumor tissue will be dissect out for biochemical investigations. The antitumor efficacy of different treatments is reported as percent increase in life span (ILS) and calculated as per the following formula:

(T/ C x 100) – 100, where T and C are the mean survival days of treated and control mice, respectively.

Glutathione estimation:-

Total GSH (TGSH) and non-protein thiol (NPSH) content will be determined using the method of Sedlak and Lindsay. Briefly, 5% homogenates of tissues will be prepared in 0.02 mol/ L EDTA, pH 4.7 in a motor-driven Teflon–pestle homogenizer. TGSH will be determined by adding 100 ml of the homogenate to 1.0 ml of 0.2 mol/L Tris-EDTA buffer, pH 8.2 and 0.9 ml of 0.02 mol/L EDTA, pH 4.7 followed by 20 μl of Elliman’s reagent (10 mmol/L DTNB in methanol). For the determination of NPSH, 500 μl of the homogenate will be precipitate with 500 μl of 10% trichloroacetic acid and centrifuged at 3000 × g for 15 min. To 800 μl of the supernatant, 1.6 ml of 0.4 mol/L, Tris-EDTA buffer, pH 8.9, will be add before the addition of 25 μl of Elliman’s reagent. After 30 min of incubation at room temperature, the reaction mixture will be centrifuge and the absorbency of supernatants will be read against a reagent blank at 412 nm using a Spectrophotometer.



Statistical analysis

The significance of difference between groups will be test using one-way ANOVA followed by post-hoc Fisher’s LSD test. P<0.05 was considered significant.



Animals Required:

12 Swiss albino mice will be used for acute toxicity studies.



100 Swiss albino mice will be used for Glutathione estimation test.



Group

Treatment

After treatment

No. of animal

I

Normal saline.

1st day

5

3rd day

5

6th day

5

12th day

5

II

1/3 of LD50 of methanolic extract of Seabuckthorn

1st day

5

3rd day

5

6th day

5

12th day

5

III

1/7 of LD50 of methanolic extract of Seabuckthorn

1st day

5

3nd day

5

6th day

5

12th day

5

IV

Cyclophosphamide

1st day

5

3rd day

5

6th day

5

12th day

5

V

Methanolic extract of Seabuckthorn + Cyclophosphamide

1st day

5

3rd day

5

6th day

5

12th day

5


Enclosure-VI
7.3 Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly.

The above study requires investigation on Swiss albino mice (112 no.) for acute toxicity study and anticancer activity.


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

The study is cleared from Ethical Committee of the institution. (Certificate enclosed).



Enclosure-VII

8. Reference:



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  2. Nicol BM, Prasad SB. The effect of Cyclophosphamide alone and in combination with ascorbic acid against murine ascites Dalton’s lymphoma. Ind J Pharmacol 2006 Aug;38(4):260-5.

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  25. Gao ZL, Gu XH, Cheng FT, Jiang FH. Effect of Sea buckthorn on liver fibrosis: A clinical study. World J Gastro enterol 2003 Jul;9(7):1615-7.

  26. Pang X. et al. Antihypertensive effect of total flavones extracted from seed residues of Hippoiphae rhamnoides L. in sucrose-fed rats. J Ethnopharmacol 2008;117:325–31.

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  32. Cao Q , Qu W , Deng Y , Zhang Z , Niu W , Pan Y . Effect of flavonoid from the seed and fruit residue of Hippophae rhamnoides L. on glycometabolism in mice. Zhong Yao Cai 2003 Oct;26(10):735-7

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