Comparative analytical profile of Benincasa hispida thumb. and Cucurbita maxima duchesne

Sheetal Sharma; Anjali Upadhyay; Pramod R Yadav; R Galib; Pradeep Kumar Prajapati

doi:10.4103/ijaim.ijaim_11_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 3 | Issue : 2 | Page : 69-77

Comparative analytical profile of Benincasa hispida thumb. and Cucurbita maxima duchesne

Sheetal Sharma, Anjali Upadhyay, Pramod R Yadav, R Galib, Pradeep Kumar Prajapati
Department of Rasa Shastra and Bhaishajya Kalpana, All India Institute of Ayurveda, New Delhi, India

Date of Submission	07-Apr-2022
Date of Decision	15-Nov-2022
Date of Acceptance	22-Nov-2022
Date of Web Publication	12-Dec-2022

Correspondence Address:
Sheetal Sharma
Department of Rasa Shastra and Bhaishajya Kalpana, All India Institute of Ayurveda, Gautampuri, Sarita Vihar, Mathura Road, New Delhi - 110 076
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijaim.ijaim_11_22

Abstract

Background: Cucurbitaceae is a family of health-promoting plants due to their compounds with beneficial effects. This is a family of about 130 genera and about 800 species. Seeds or fruit parts of some cucurbits are reported to possess many therapeutic activities. Benincasa hispida Thumb. familiarly known as Kushmanda and Cucurbita maxima Duchesne as Kashiphala are two varieties of Cucurbitaceae family. Thus, comparative analysis of both these varieties is done to evaluate the analytical profile.
Aim: To evaluate the analytical profile of B. hispida and C. maxima.
Materials and Methods: Fresh juice, semi-solid, dried powder of both the samples were prepared in the laboratory, and comparative analytical profiles were developed by following standard guidelines of Ayurvedic Pharmacopoeia of India (API).
Results and Conclusion: The organoleptic parameters include changes in color, taste, and odor of both samples. In physico-chemical parameters, slight changes in loss on drying, Ph, and Total solid content was mentioned. Total ash value and extractive values have found within the limit as mentioned in API. Phyto-chemical analysis shows the presence of alkaloids, carbohydrates, reducing sugars, flavonoids, and steroids in both varieties. Powder microscopy of both samples showed parenchymatous cells, xylem fibers, xylem tracheid's, epidermal cells, and starch grains which were found sparse in B. hispida and dense in C. maxima. High-performance thin-layer chromatography revealed similarity of 3,4,3 bands at 254 nm and 4,3,4 bands at 366 nm ultraviolet detection in methanolic extract, aqueous, hydro-alcoholic extract, respectively, and fourier-transform infrared spectroscopy study of B. hispida and C. maxima showed the presence of amines, methylene group and alcohols, phenols, and methylene group, respectively.

Keywords: Benincasa hispida, Cucurbita maxima, fourier-transform infrared spectroscopy, high-performance thin-layer chromatography, phytochemical screening

How to cite this article:
Sharma S, Upadhyay A, Yadav PR, Galib R, Prajapati PK. Comparative analytical profile of Benincasa hispida thumb. and Cucurbita maxima duchesne. Indian J Ayurveda lntegr Med 2022;3:69-77

How to cite this URL:
Sharma S, Upadhyay A, Yadav PR, Galib R, Prajapati PK. Comparative analytical profile of Benincasa hispida thumb. and Cucurbita maxima duchesne. Indian J Ayurveda lntegr Med [serial online] 2022 [cited 2023 Jun 6];3:69-77. Available from: http://www.ijaim.in/text.asp?2022/3/2/69/363106

Introduction

Cucurbitaceae have largely worldwide distributions but occur mostly in the tropical regions. Benincasa hispida Thumb. and Cucurbita maxima D. are the two varieties of Cucurbitaceae family taken for this study. Seeds or fruit parts of some cucurbits are reported to possess purgatives, emetics and anti-helminthic properties due to secondary metabolite cucurbitacin content.^[1] B. hispida (Kushmanda) has been used as a chief ingredient in the formulation named Kushmanda Rasayana which is having immense potential in curing many ailments. Acharya Charaka has mentioned that kushmanda is beneficial in eliminating all doshas from body.^[2] In Shusruta Samhita, the therapeutic efficacy of Kushamnda has been ascribed for psychological disorders.^[3] Acharya Vagbhata has described Kushmanda as the best creeper fruit.^[4] Being described with a wide range of therapeutic utilities its botanical identification becomes mandatory. In the scientific literature B. hispida and C. maxima has been reported to be of same family, i.e., Cucurbitacea, but there is ambiguity between the species to considered it Kushmanda as the quality control of Indian medicinal plants consider C. maxima as Kushmanda, whereas other classics have mentioned B. hispida as Kushmanda. Therefore, the present comparative study has been conducted to explore the differences between the two species at the analytical level.

Phytochemical analysis showed that the major constituents of B. hispida Linn. fruits are volatile oils, flavonoids, glycosides, saccharides, proteins, carotenes, vitamins, minerals, ß-sitosterin, and uronic acid.^[5] Methanolic extract of B. hispida has been reported to exhibit mast stabilizing effect and potential inhibitory effect on histamine release induced by antigen-antibody reaction owing to the presence of two major triterpenes namely alonusenol and multiflorenol.^[6]

C. maxima D. contains carbohydrates, proteins, fat, fiber and amino acids, tocopherols, beta-carotenoids, beta-Cryptoxanthin.^[7],[8] Study reported about C. maxima exhibits important physiological properties such as wound healing, tumor growth inhibition, hypoglycaemic effects, and immune-modulating.^[9] The study revealed the action of seeds of C. maxima has an action on bronchitis.^[10] C. maxima fruits and seeds have been studied for the last decade due to their developed ethnopharmacological applications in the treatment of parasitical diseases.^[11

B. hispida is said to be Kushmanda in classical texts like Dravyaguna Vigyaniyama Part-2 authored by Acharaya Priyavrta Sharma^[12] and C. maxima is mentioned as Kushmanda in Quality Standards of Indian Medicinal Plants of ICMR in the pictorial representation.

B. hispida and C. maxima are being used in the name of Kushmanda, but analytical profiles of both the samples are not available. Hence, fresh juice, semi-solid, and powder form of both the samples have been taken for the present study.

Materials and Methods

Fruits of B. hispida (Voucher Specimen no.-RRDR/AIIA/10) [Figure 1]a and C. maxima (Voucher Specimen no.-RRDR/AIIA/103) [Figure 2]a were collected in July 2021 and authenticated by Pharmacognosy Laboratory of AIIA Sarita vihar, New Delhi. In [Figure 1]b and [Figure 2]b pictorial representation of both samples along with creeper is shown. [Figure 1]c and [Figure 2]c representing cut pieces of both the fruits, respectively. [Figure 1]d and [Figure 2]d representing semisolid extract of both the fruits respectively. All chemicals (AR grade) and thin-layer chromatography (TLC) plates were purchased from E. Merck Pvt. Ltd.(Mumbai, India).

Figure 1: (a) Benincasa hispida fruit. (b) Benincasa hispida cut pieces. (c) Benincasa hispida Swarasa. (d) Benincasa hispida Semi-solid

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Figure 2: (a) Cucurbita maxima fruit. (b) Cucurbita maxima Cut piece. (c) Cucurbita maxima Swarasa. (d) Cucurbita maxima Semi-solid

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Organoleptic and physico-chemical evaluation

All the organoleptic tests color, odor, taste, etc., of the B. hispida and C. maxima were executed. Physicochemical parameters including moisture content (Loss on Drying),^[13] pH, total ash,^[14] acid insoluble ash,^[15] water-soluble extractive value^[16] and alcohol soluble extractive values^[17] of both samples have been evaluated and placed in [Table 1], following Ayurvedic Pharmacopoeia of India.^[18]

Table 1: Organoleptic parameters of Benincasa hispida and Cucurbita maxima Swarasa

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Phytochemical analysis

The prepared alcoholic extracts of the drug were subjected to initial phytochemical screening following standard methods.^[19]

Powder microscopy

Shade dried cut pieces of fruits of B. hispida and C. maxima powdered with the help of grinder and passed through 60 no. sieve to obtain coarse powder. These powders [Figure 3]a and [Figure 3]b were subjected to powder microscopy as per the standard procedures.^{[19],[20],[21]}

Figure 3: (a) Dry powder of Benincasa hispida, (b) Dry powder of Cucurbita maxima

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High-performance thin-layer chromatography sample preparation

The plant parts were dried under a gentle stream of air in the laboratory till no loss in weight (temperature 30°C + 2°C and relative humidity 50 + 5%) and powdered in an electric grinder. Conventional extraction of C. maxima fruit and B. hispida fruits were performed separately at room temperature (28°C ± 3°C) with a variety of polar solvents ranging from methanol, mixture of methanol: water (50:50) and water. Each drug (10 g) was extracted three times (3 × 50 mL) for 18 h separately with each of the above-mentioned solvents. Each extract was filtered by using Whatman filter paper no. 1 and the solvents were removed under vacuum at 50°C, separately and concentrated up to 10 mL to get the sample solution of 100 mg/mL. 5 μL of each sample was applied separately to the high-performance TLC (HPTLC) plate for the development of fingerprints.^[22]

High-performance thin-layer chromatography method

HPTLC was performed on 20 cm × 10 cm TLC plates pre-coated with 0.25 μm thin layers of silica gel 60 F254 (E. Merck). B. hispida and C. maxima extracted in methanol (1:10) and then this were applied separately in triplicate manner on the plates as bands 10 mm wide by use of a Linomat-IV applicator (CAMAG, Switzerland) fitted with a 100 μL syringe (Hamilton, Switzerland). The application positions X and Y were both 10 mm, to avoid edge effects. Linear ascending development to a distance of 80 mm with Benzene: Ethyl acetate (9: 1 (v/v)) and as mobile phase for all extracts were performed in a twin-trough glass chamber (20 cm × 10 cm) previously saturated with vapors of mobile phase for 20 min. The fresh plate in visible light is shown in [Figure 4]a. This plate was dried in air and visualized under λ 254 nm and λ 366 nm for ultra violet detection and taken the fingerprints as evident in [Figure 4]b and [Figure 4]c. Peak value of HPTLC data is shown in [Figure 5]a and [Figure 5]b.

Figure 4: (a) Visible-light, (b) 254 nm. (c) 366 nm

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Figure 5: (a) HPTLC fingerprints at 254 nm. (b) HPTLC fingerprints at 366 nm. HPTLC: High-performance thin-layer chromatography

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Track position from left to right

Track A-B. hispida aqueous extract, Track B-C. maxima aqueous extract, Track C-B. hispida methanolic extract, Track D-C. maxima methanolic extract, Track E-B. hispida hydroalcoholic extract, Track F-C. maxima hydroalcoholic extract.

FTIR analysis

1 mg powder (mesh size #120) sample and potassium bromide (KBr) were mixed at a ratio of 1:100 and pellets were prepared applying the same pressure to keep the thickness of the pellet constant. Utmost care was taken to obtain the same concentration of sample each time in every pellet. FTIR spectra were recorded in the 400–2000 cm⁻¹ spectral region with an FTIR spectrophotometer (Spectrum RX-I; PerkinElmer). The spectra were collected at a resolution of 4 cm⁻¹. Each spectrum is an average of 10 scans.^[23]

Results

Organoleptic and Physico-chemical parameters of B. hispida and C. maxima Swarasa, Semi-solid and powder have been depicted in [Table 1],[Table 2],[Table 3],[Table 4],[Table 5],[Table 6] respectively. The physico-chemical parameters included as moisture content, ash values including total ash value, acid insoluble ash value, extractive values such as water soluble and alcohol soluble extractive values. The detection of chemicals present in our sample by phyto-chemical screening is given in [Table 7] and chromatographic analysis by HPTLC method is given in [Table 8].{Table 1}

Table 2: Physicochemical parameters of Benincasa hispida and Cucurbita maxima Swarasa

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Table 3: Organoleptic parameters of Benincasa hispida semisolid and Cucurbita maxima semisolid

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Table 4: Physicochemical parameters of Benincasa hispida semisolid and Cucurbita maxima semisolid

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Table 5: Organoleptic parameters of Benincasa hispida powder and Cucurbita maxima powder

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Table 6: Physicochemical parameters of Benincasa hispida powder and Cucurbita maxima powder

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Table 7: Phytochemical screening of both powder samples

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Table 8: High-performance thin-layer chromatography profile of samples

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Powder microscopy of both samples revealed different structures given below.

The dried powder of B. hispida was mounted in glycerine and conc. Hcl showed lignified
Parenchymatous cells [Figure 6]a, crystals of calcium [Figure 6]b. After staining with Phloroglucinol sparse starch patches [Figure 6]c found. Also stained with Safranin found Xylem tracheids [Figure 6]d
The dried powder of C. maxima mounted in glycerine and conc. HCL found Xylem tracheids [Figure 7]a Starch grains [Figure 7]b, Lignified parenchyma cells [Figure 7]c Stone cells [Figure 7]d.

Figure 6: (a) HCL 40 × 2 lignified parenchymatous cells. (b) HCL 40 × 4 crystals of calcium. (c) Phloroglucinol 10 × 1 StarchPatches. (d) Safranin 40 × 1 xylem tracheid's

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Figure 7: (a) HCL 40 × Xylem Tracheids. (b) HCL 40 × 1 Starch Grains. (c) HCL 40 × 2 Lignified Parenchyma Cells. (d) Safranin 40 × 1 Stone Cells

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B. hispida Thumb
C. Maxima D.

FTIR

The spectrum of B. hispida fruit powder shows bands at 3413.55, 2928.43, 2345.16, 1637.13, 1384.72, 1237.11, 1056.05, 778.78, 527.15 cm⁻¹. The IR absorbance corresponding to a-aromatic 1° amine NH Stretch is observed at 3413.55 cm⁻¹. The band at 2928.43 cm⁻¹ is due to methylene C-4 asym. Stretch. FTIR Spectra of both the samples is given in [Figure 8]a and [Figure 8]b, respectively.

Figure 8: (a) FTIR peak absorbance Benincasa hispida powder pellet. (b) FTIR peak absorbance of Cucurbita maxima fruit powder pellet. FTIR: Fourier-Transform Infrared Spectroscopy

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Discussion

Organoleptic parameters play a crucial role in patient compliance for the drug and to evaluate its quality because desired characteristics will appear only when standard operating procedures and quality standard where adopted. In [Table 3] and [Table 5], there is no such difference between powder and semisolid of B. hispida but in C. maxima semi-solid sample color is caramel, whereas in powder stage, it converts to dark brown color. In [Table 2], the physical parameters such as pH were determined to avoid gastric irritation. The pH of swarasa of B. hispida and C. maxima was found to be acidic and basic in nature, respectively, but the dry powder of C. maxima was found to be acidic in nature. It is possibly because of evaporation of constituents which were alkaline in nature. Besides, in Ayurveda Kushmanda has been indicated to be used in fresh state and it has been observed in the present study that Ph of both the species differ in their fresh state which may lead to the difference in their pharmacological property and site of absorption.

The moisture content was determined to find out any increase in weight caused by moisture absorption. Since ashing process involves oxidation of components of product, an increase in ash value indicates contamination, substitution, and adulteration. The total ash value is an indicative of total amount of inorganic material after complete incineration and the acid insoluble ash value obtained is an indicative of silicate impurities, which might have aroused due to improper washing of crude drugs. Both the ash values obtained were found to be within the standard limits. The extractive values namely water-soluble and alcohol soluble indicate the amount of active constituent in given amount of plant material when extracted with respective solvents, a lower value compared to standard value indicates presence of exhausted material. In the present study, both the sample water extractive values were found to be less than standard value and alcohol extractive value is more than the standard values. Qualitative tests are used to detect the presence of functional groups, which play a very important role in the expression of biological activity. The present study reveals the presence of alkaloids and steroids in both extract alcoholic and water soluble. Carbohydrates and flavonoids were also seen in alcoholic extract of both samples [Table 7].

HPTLC fingerprints of methanolic extracts of fruits of B. hispida and C. maxima showed 15 and 14 bands under ultraviolet (UV) detection at 254 nm, respectively, out of which only three spots at R_f 0.04, 0.25, and 0.41 were found similar in both drugs. Under 366 nm UV detection, phytochemical fingerprints of methanolic extracts of fruits of B. hispida and C. maxima showed 5 and 6 bands respectively, out of which four bands at R_f 0.06, 0.22, 0.63 were found similar in both drugs [Table 8].

HPTLC fingerprints of aqueous extracts of fruits of B. hispida and C. maxima showed 15 and 11 bands under UV detection at 254 nm, respectively, out of which Rf 0.07, 0.21, 0.30, 0.67, and 0.95 were found similar in both drugs. Under 366 nm UV detection, phytochemical fingerprints of aqueous extracts of fruits of B. hispida and C. maxima showed 4 and 3 bands, respectively, out of which no R_f found similar in both drugs [Table 8].

HPTLC fingerprints of hydro-alcoholic extracts of fruits of B. hispida and C. maxima showed 9 and 15 bands under UV detection at 254 nm, respectively, out of which R_f 0.03, 0.25, 0.52, and 0.65 were found similar in both drugs. Under 366 nm UV detection, phytochemical fingerprints of aqueous extracts of fruits of B. hispida and C. maxima showed 3 and 4 bands, respectively, out of which R_f 0.06, 0.30, and 0.60 found similar in both drugs.

B. hispida and C. maxima have a mixture of chemical constituents so interpretation of spectrum is done up to the level of possible chemical interaction and thus functional group in it. [Figure 8]a shows that the IR spectrum of B. hispida fruit powder shows bands at 3413.55, 2928.43, 2345.16, 1637.13, 1384.72, 1237.11, 1056.05, 778.78, and 527.15 cm⁻¹. In B. hispida fruit, the broad band at 3413.55 cm⁻¹ belongs to the NH stretching frequency that may be core of aromatic primary amine compound, 2928.43 cm⁻¹ C-H bond asym. Stretch core of methylene group 1637.13 cm⁻¹ frequency shows unsaturated bond, in 1237.11 cm⁻¹ shows C-C vibrations. 1056.05, 778.78 and 527.15 shows C-F, C-Cl and C-I Stretch [Table 9]. [Figure 8]b shows that in C. Maxima broad peak observed at 3402.32 cm⁻¹ frequency shows O H stretch H bonded because of alcohols, phenols, 2930.11 cm⁻¹ belongs to C-H Asymmetrical Stretch/Symmetrical Stretch may be of methylene group, 2345.95 cm⁻¹ shows C = C and in can be found in alkynes. 1055.65 cm⁻¹ and 586.4 cm⁻¹ show C-F and C-I stretch bond [Table 10].

Table 9: FTIR peak value and functional group of Benincasa hispida fruit powder

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Table 10: FTIR peak value and functional group of Cucurbita maxima

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The phytochemical analysis of samples revealed the presence of alkaloids, carbohydrates, and reducing sugars. The presence of alkaloids and sterols contribute to its anti-inflammatory activities.^[24] Carbohydrates and reducing sugars may be responsible for the nutritive potential of the Kushmanda.^[25] These therapeutic properties correspond to the rasayana effect mentioned in Ayurveda.

The present study reveals significant differences in the microscopic examination and presence of functional group in B. hispida and C. maxima. These findings will help in identifications of B. hispida and provide standard values for certain quality parameter pertaining to Kushmanda which can be used to identify any type of adulteration.

The observations infer that there is a difference in between two samples. As Kushmanda is preferred in therapeutic use, further advanced instrumental analysis appears essential to evaluate and estimate actual therapeutic moiety responsible for its therapeutic action.

Conclusion

On the basis of parameters such as organoleptic parameters, physico-chemical parameters, HPTLC, FTIR of B. hispida and C. maxima; it can be concluded that there is difference of chemical composition in both the herbs. So for, therapeutic efficacy B. hispida should be used for the preparation of Ayurvedic formulations. B. hispida is a commonly used melon having many names such as wax gourd, white melon, and white pumpkin which has been found in classical texts for its medicinal properties. It is employed as main ingredient of Kushmanda Rasayana in Ayurvedic system of medicine. C. maxima which is also known as red gourd has been mentioned in classical nighantus with their medicinal properties. In quality standards of Indian Medicinal Plants, B. hispida (Thumb.) has been mentioned but pictorial representation is made for C. Maxima.^[26] The phyto-chemical screening has revealed the presence of alkaloids, carbohydrates, reducing sugar, flavonoids, and steroids in both samples have found almost same but FTIR and HPTLC have shown chemical variations. This will be considerable for further studies. Till the date, data have been collected for one sample for both the herbs B. hispida and C. maxima which was collected from one region only. Triplicate sample of both the herbs has been run for HPTLC. Rf values which are found the same have been highlighted and difference in Rf values shows the presence of difference of chemical constituents in both samples which will be further evaluated with advanced instrumental techniques and help of markers if available.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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