A study for Quality Assessment of the Dried Seeds of Benincasa hispida

Hayat M. Mukhtar*, Deepika Vashishth, Babar Ali, Rupinder Kaur

Natural Product Research Laboratory, Department of Pharmacognosy and Phytochemistry, Shaheed Bhagat Singh College of Pharmacy, Patti, Amritsar, Punjab- 143416

ABSTRACT:

This specification covers the standardization and preliminary phytochemical investigation of the plant Benincasa hispida (Cucurbitaceae). It is widely used as antiulcer, anti-angiogenic, bronchodilator, in opioid withdrawal benefit, anti-diabetic and in mental disability. The plant material was subjected to pharmacognostical studies like macroscopic and microscopic studies (powder microscopy, micrometry, histochemical tests and transverse section). The microscopic characters revealed the presence of: Seeds [lignified trichomes found in groups (17.28µ,1.62µ), transparent fibers (70.20µ, 1.35µ), round shaped oil cells (9.45µ) found in thin walled parenchyma cells, parenchyma cells with wrinkled walls and lignified spiral vessels (52.38µ, 1.48µ)] mesocarp [lignified covering trichomes (27.43µ, 1.73µ) fibers (34.10µ, 2.22µ), lignified parenchyma cells (14.58) and vascular bundles]. Dried plant material was subjected to various physiochemical parameters like hot extraction, ash values, cold maceration, thin layer chromatography with different solvents and at different wavelengths, fluorescent analysis, loss on drying, foaming index, swelling index, successive extraction. The preliminary phytochemical investigation revealed the presence of amino acids, triterpenoids, tannins, carbohydrates. These parameters can be utilized for quick identification of the Benincasa hispida and are particularly useful in powdered form.

KEYWORDS: Benincasa hispida, powdered seeds, microscopy, amino acids, triterpenoids.

INTRODUCTION:

Khusmand consists of the dried pieces of fruits of Benincasa hispida (Thunb.) Cogn. (Family Cucurbitaceae), an extensive trailing or climbing herb cultivated throughout the plains of India and on the hills upto 1200 m altitude, as a vegetable¹. The fruits are large broadly cylindrical covered with whitish wax thoroughly. The seeds are yellowish cream about 1-1.5 cm length and 0.5 to 0.07 cm width having fine wrinkles. The leaves are green, large and heart shaped almost 10-15 cm in diameter. The seeds contain stable oil and fruit 96% moisture. Fruits of this plant are traditionally used as a laxative, diuretic, tonic, aphrodisiac, cardiotonic, in urinary calculi, blood disease, insanity, epilepsy and also in cases of jaundice, dyspepsia, fever, menstrual disorders, used to improve metabolism and provides general strength. The fruit possess alternative and styptic properties and is populary known as valuable antimercurial. The fruits and seeds are used for medicinal purpose. Externally, the pulp of fruit is applied on wounds and burns to alleviate the burning sensation. The seeds mashed with water, serve the same purpose. In headache the seed oil is massaged for relief². Detection of sitosterol, lupeol, triacontanol, mannitol, arginine, aspartic acid, glutamic acid asparagines, glutamine, praline, hydroxyproline, isoleucine, cysteine, L-leucine, glucose and rhamnose in fruits by TLC³

.

The crop can be grown in plains in late winter by adopting polyhouse technology⁴. Benincasa hispida is helpful in fire burns and mental disabilities. It specifically relieves memory loss and other mental disorders⁵. It may prove beneficial in reproductive disorders⁶.

MATERIAL AND METHODS:

Plant material:

The seeds of Benincasa hispida were collected from Hoshiarpur (Punjab) in the month of August. The seeds were identified and authenticated by botanist, Dr. Adarsh Pal Vig, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar. Its voucher number (1511/Hbr) specimen was deposited in the same department for reference.

Processing of Plant material:

After authentication, seeds were dried at room temperature until they were free from the moisture and subjected to physical evaluation for different parameters.

Reagents:

All the reagents were of Analytical grade and purchased from S.D Fine- Chem. Ltd., Mumbai, India.

Methods:

The seeds were standardized on the basis of quality parameters as per the WHO guidelines⁷. The organoleptic characters including colour, odour, taste and external features of seeds were observed and the results were recorded. The microscopy of seeds was also studied which included powder microscopy, micrometry, transverse section and histochemical test. The extractive values were determined by successively starting from petroleum ether, benzene, chloroform, ethyl acetate, acetone and methanol⁸ by using soxhlet extraction apparatus. The dried extractive values were obtained after evaporation of solvent under reduced pressure using vaccum rotary evaporator. The behaviour of the powdered seeds with different chemical reagents were studied and fluorescence characters were also observed under ultraviolet and visible light⁹ (compared with nerolac synthetic and satin enamel colour card).The pH of seeds extract was measured using a pH meter of glass electrode, pH fundamentally represents the value of hydrogen ion activity in solutions. Preliminary phytochemical tests of different extracts of seeds were performed with specific reagents ^{10, 11}

RESULTS:

The dried seeds of Benincasa hispida were subjected to standard procedures according to WHO Guidelines for the determination of various physicohemical parameters. The following parameters were determined:

Macroscopic characters:

This included morphological studies of seeds (Fig: 1) such as colour, size, odour, taste, surface (Table1).

Table: 1 Organoleptic characters of the seeds of Benincasa hispida

Organoleptic Characters	Observations
Colour	Yellowish cream
Odour	Odourless
Taste	Nutty
Size	1.0-1.5cm length, 0.05 -0.07 cm width
Shape	Simple

Fig1. Benincasa hispida seed

Microscopic characters

This parameter included micrometry (Table 2), histochemical test (Table 3), transverse section (Fig: 2) of seeds.

Table2: Micrometry of seeds of Benincasa hispida

Microscopic characters	Size (µ)
Trichome	17.28, 1.62
Fiber	70.20, 1.35
Oil cell	9.45
Vessels	52.38, 1.48

Fig: 2 T.S of seed of Benincasa hispida

Ash Values

The determination of ash value is meant for detecting low-grade drugs, exhausted drugs, sandy or earthy matter. The results of ash values were recorded in (Table 4).

Extractive Values

This method determines the amount of active constituents extracted with solvents from a given amount of medicinal plant material. It is employed for materials for which as yet no suitable chemical or biological assay exists. The air dried, accurately weighed drug was treated with solvents: petroleum ether, benzene, chloroform, ethyl acetate and methanol. The values were recorded in (Table 5).

Table: 3 Histochemical test of T.S of seeds of Benincasa hispida.

Reagents	Colour	Constituents	Tissue	Degree ofIntensity
Phloroglucinol + HCl	Dark pink	Lignin	Seed covering	+++
Iodinated zinc chloride + Iodine + H2SO4	Blue to blue violet	Cellulose cell walls	Epicarp	++
Iodine/ethanolTS +trinitrophenol + mercuric nitrate	Brick red	Aleuron grain	Endothelium and endosperm	++
Potassium hydroxide	Red	Hydroxyl anthraquinones	Pericarp	+
1 napthol TS + H2SO4	Brownish red	Inulin	_	_
Iodine + glyceroethanol	Redish blue	Starch	Epidermis	+
Ferric chloride	Bluish black	Tannin	Seed covering	++
Sudan Red solution		Oil globules	Endosperm	+++
Millon reagent	Light yellow	Amino acid	Endocarp	+

Note: +++High, ++Moderate, +Slight, -Nil.

Moisture content

Karl Fisher and loss on drying methods were used to determined the moisture content of seeds. Moisture content level helps to reduce the chances of microbial contamination (Table 6).

Swelling index:

1g of powdered drug was dipped in 25 ml of water in a 25 ml of glass stoppered measuring cylinder. Swelling index is high in mucilaginous drugs (Table 7).

Foaming index:

The foaming ability of an aqueous decoction of plant material and their extract is measured in term of foaming index (Table 7).

pH value

pH is measured using a pH meter of glass electrode. pH fundamentally represents the value of hydrogen ion activity in solutions¹². The values are recorded in (Table 7).

Fluorescence analysis

The powdered drug was examined under ordinary light, short UV (254 nm) and long UV (366 nm) light using different solvents and compared with nerolac synthetic and satin enamel colour reference card. The results were recorded in (Table 8).

Table: 4 Ash values of seeds of Benincasa hispida

Parameters	Values obtained (% w/w)
Total ash	4
Water soluble ash	1
Acid insoluble ash	0.5
Sulphated ash	3

Table: 5 Extractive values of seeds of Benincasa hispida

Solvents	Seed
	SEV (% w/w)	CMV (% w/w)
Petroleum ether	18	29
Benzene	1.2	28
Chloroform	1.8	28
Ethyl acetate	1.6	28
Acetone	2.4	25
Methanol	4	12

Abbr. SEV: Successive extractive value CMV: Cold maceration value

Table: 6 Moisture content of Benincasa hispida seeds with different methods

Method	Moisture content (%)
Karl Fisher titration	5.8
Loss on drying	5.6

Table: 7 Parameters for seeds of Benincasa hispida:

Swelling index of seeds in ml	1.5
Foaming index of seeds	≥100
pH value of seeds	6.22

Table: 8 Fluoresence analysis of powdered seeds of Benincasa hispida with various chemical reagents under U.V. (Compared with nerolac synthetic and satin enamel)

Solvents	Visible light	UV (254nm)	UV(366nm)
Drug Powder as such	Fantan	Lime bright	Florida everglade
Drug + Conc. H2SO4	Black	Black	Black
Drug + Conc. H2SO4 + Distilled water	A.D grey	Pista	Espana
Drug + Conc. HCl	Sugar cookie	Water grey	Florida everglade
Drug + Conc. HCl + Distilled water	Nanette	Shadow lime	Cherry
Drug + Conc. HNO3	Pumper nickel	Cinnamon tea	Cherry
Drug + Conc. HNO3 + Distilled water	Royal ivory	Lime bright	Po red
Drug + Methanol	Royal ivory	Gucci	Terracotta
Drug + Chloroform	Festive	Sugar cookie	Burnt brick
Drug +Petroleum ether	Espana	Lime peel	Sugar creek
Drug + Ferric Chloride	Mantego grey	Pista	Brown
Drug + Picric acid	Nanette	Lime peel	Black
Drug + 10% Sodium hydroxide	Lime yellow	Tata mimosa	Leaf brown
Drug + Ammonia solution	Pale cream	Tata mimosa	Brown
Drug+Distilled water	Golden wheat	Leaf peel	Sugar creek
Drug+ Acetic acid	Espana	Gucci	Terracotta
Drug+ Ethanol	Golden wheat	Pista	Leaf brown
Drug+ 5% Iodine	Brown	Deep green	Black

Preliminary phytochemical screening

The plant material was subjected to preliminary phytochemical screening for the detection of various plant constituents. The extracts obtained from successive solvent

extraction were subjected to qualitative test for the identification of various plant constituents like alkaloids, carbohydrates, glycosides, phenolic compounds, flavonoids, proteins and amino acids, saponins, steroids, sterols, acidic compounds, mucilage and resins. The results were recorded in (Table 9).

Table: 9 Preliminary phytochemical screening of seeds of Benincasa hispida.

Phyto constituents	PEE	BE	CE	EAE	ME
Alkaloids	-	-	-	-	-
Glycosides	-	-	-	-	-
Proteins and Amino acids	++	+	-	-	-
Carbohydrates	++	+	+	-	+
Tannins	-	-	+	++	+
Fats and Fixed oils	+++	+	-	-	-
Saponins	-	-	-	-	-
Steroids	+	-	++	-	-
Flavonoids	+	-	-	-	+
Triterpenoids	+	+	+	-	+

Abrr. PEE: Petroleum ether extract BE: Benzene extract CE: Chloroform extract EAE: Ethyl acetate extract ME: Methanol extract

DISCUSSION:

Generated data can be used for determining correct identity and purity of plant parts and for the detection of adulteration. Botanical authentication and physicochemical parameters will give an idea about the quality of drug. All these parameters which are being reported could be useful in identification of distinctive features of the drug. From the preliminary phytochemical study, it was concluded that Benincasa hispida seeds contains the reported phtoconstituents. Hence, detailed screening may be done to isolate the active constituents so that it may be scientifically proved to access the pharmacological responses of the plant to ascertain its folklore uses.

ACKNOWLEDGEMENTS:

The authors wish to thank SBS College of Pharmacy, Patti, for providing necessary facilities.

REFERENCES:

1. Ayurvedic Pharmacopeia of India. Government of India, Ministry of Health and Family Welfare, Department of Ayush. part- 1 vol. 4: pp. 62-63.

2. Kritikar KR and Basu BD. Indian Medicinal Plants. Sri Satguru Publications, Delhi. 2005; 2^nd ed: pp. 1127-1128.

3. Yoshizumi S, Murakami T, Kadoya M, Matsuda H, Yamahara J and Yoshikawa M. Histamine release inhibitors from wax gourd, the fruits of Benincasa hispida Cogn., Medicinal foodstuffs XI. 118; 1998:188–92.

4. The Wealth of India. Dictionary of Indian Raw Materials and Industrial Products, CSIR, New Delhi. 2003; vol 4: pp. 133-134.

5. Nilangekar P and Murthy VPC. Kushmandu – a wonderful drug, Sachitra Ayurved, Hyderabad, A.P., India. 49(3); 1996: 171-173.

6. Sakya TR and Prasad RB. Nepal Biological studies for abortifient activity of Benincasa hispida, NepJOL, 7 (special issue); 1979: 77-79.

7. WHO guidelines. Library cataloguing in publication data, Quality Control Methods for Medicinal Plant Material, WHO Geneva, Delhi. 1998; pp. 1-110.

8. Mukherjee K Pulok. Quality Control of Herbal Drugs, An Approach to Evaluation of Botanicals, published by Business Horizons. New Delhi. 1st ed: 2002; pp. 446-447.

9. Raghunanthan K and Mitra R. Pharmacognosy of Indigenous Drugs, Central Council for Research in Ayurveda and Siddha, New Delhi. 1; 1982: 575-576.

10. Trease GE and Evans WC. Pharmacognosy, Elsevier Publications. 15th ed: 1985; p. 30.

11. Tyler VE Brady LR and Robbers JE. Pharmacognosy, Lea and Febiger Publication, Philadelphia. 9th ed: 1985; pp.527-528.

12. Indian Pharmacopeia. Ministry of Health and Family Welfare, New Delhi. vol. 2: 1996; p. A-95.

Received on 10.05.2012

Modified on 25.05.2012

Accepted on 30.05.2012

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 4(4): July- August 2012, 201-204