Tribulus terrestris L. fruits: An extensive Pharmacognostical and Phytochemical Quality Assesment using UV-spectrophotometer

 

Sonia, Singh Sumitra*

Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology,

Hisar – 125001, Haryana, India.

 

ABSTRACT:

The qualitative and quantitative studies were performed on Tribulus terrestris L. fruits to ascertain the quality parameters. T. terrestris L. fruits were analyzed for macroscopical, microscopical studies, physico-chemical parameters, fluorescence studies, elemental analysis, preliminary phytochemical screening, total phenolic, flavonoid, tannin, saponin and alkaloid content. For macroscopy fresh fruits were used. n- hexane and hydroalcoholic extracts were used for preliminary phytochemical screening and quantitative estimation of total phenolic content, flavonoid content, tannin content, total saponin, alkaloid content. For quantitative estimation UV- spectrophotometer was used at required wavelength. The colour of fresh fruits was green while dried was light brown. Fruits were globose in shape. Odour was slightly aromatic and taste was slightly bitter and astringent. Transverse section of fruit showed five cocci filled with fixed oil cells while powder microscopy showed numerous trichomes, lignified and nonlignified fibers, collenchyma, endosperm cells containing fixed oil, cortex cells, xylem vessels etc. Elemental analysis was performed using Atomic Absorption Spectroscopy and showed heavy metals like mercury, arsenic, lead and cadmium etc. were within limits. Preliminary phytochemical studies revealed the presence of carbohydrates, alkaloids, glycosides, flavonoids, sterols, phenolic and tannins compounds. These studies will help to establish quality standards, purity and sample identification of T. terrestris L. fruits.

 

 

INTRODUCTION:

Pharmacognostical standardization is an efficient tool to establish quality control parameters and helps to assure the authentication of plants. These studies also ensure reproducible quality and prevents adulteration of plant material and herbal products in trade^1,2. Tribulus terrestris L. variety is also known as mitha (sweet) gokhru. The genus Tribulus, family Zygophyllaceae and section Terrestris L. it consists of 25 species in the world^3-6. TT is an annual herb about 30–70 cm high. It has pinnate leaves of unequal length, yellow flowers and characteristic stellate shaped carpel fruits. This plant is commonly known as caltrops or devil’s thorn and gokhru in Hindi⁷.

 

T. cistoides, T. longipetalus, T. terrestrisand T. zeyheri are believed weeds⁸. T. terrestris is a tropical plant distributed throughout India and a natural herb used for its medicinal effects around the world. It has a 5,000-year-old history of medicinal use in India and traditionally it has been used forboosting hormone production in men and women^9,10. T. terrestris is used for treating cutaneous pruritus, edema, inflammation and tracheitis¹¹. The beneficial effects of its fruits, such as, cooling, diuretic and tonic effects, contribute to its wide use in painful micturition, calculus affections and other urinary disorders⁷.

 

The aim of present study was to perform pharmacognostical standardization, spectrophotometric quantification of phenolic, flavonoid, tannin, saponin and alkaloid contents of T. terrestris fruits. For the standardization and quality assurance purpose, the following three traits must be verified: authenticity, purity and assays. Hence, we make an attempt for the standardization of T. terrestris fruits by carrying out its pharmacognostical studies, physico-chemical parameters. In addition, the quantification of total phenolic, flavonoid, tannin, saponin and alkaloid content were determined.

 

MATERIALS AND METHODS:

Plant material:

Tribulus terristeris L.was procured from open fields of Karnal, Haryana in month of July, 2016. The plant was authenticated by Dr. Anjula Pandey, Principal scientist, National Herbarium of cultivated plants, NBGPR, New Delhi, vide reference no. NHCP/NBGPR/2016-11 dated 19.08.2016. The plant was identified as Tribulus terristeris L. and Family Zygophyllaceae. The fruit of plant was selected for the present study. A voucher specimen of the same has been retained in the Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar for future reference. Shade dried and pulverized fruits of the plant were used for the various studies.

 

Macroscopical studies:

The macroscopical studies of T. territeris were performed by visual examination. The macroscopical characteristics like color, odour, taste, shape, size, texture and surface characteristics were determined.

 

Microscopical studies:

Microscopical study of fruit in entire form and in powdered form was performed for histological evaluation¹². Slides were observed under light microscope (Carl Zeiss Primo star, Germany).

 

Physicochemical parameters:

Physicochemical parameters of the fruit powder were studied using standard procedures. These parameters include foreign organic matter, loss on drying, extractive values, ash values, swelling index and foaming index^{12, 13,14,15}.

 

Fluorescence analysis:

A small quantity powdered T. terrestris fruits was subjected to fluorescence analysis in visible/ daylight and UV light (254nm and 365nm)^16,17,18,19.

 

Elemental analysis:

Elemental analysis of T. terrestris fruit powder was done using nitric-perchloric acid digestion method using the procedure recommended by the AOAC (1990)^20,21.

 

The samples obtained after filtration were analyzed in Atomic Absorption Spectroscopy (AAS) (GBC 932 plus). The instrument was calibrated by using the standard solutions of Pb, Cd, Cu, As, Hg, Co, Mn, Fe, K, Ca and Mg at various wavelengths 217.0, 228.8, 324.7, 193.7, 253.7, 240.7, 279.5, 248.3, 766.5, 422.7 and 285.2nm respectively. Then, the standard calibration curves of these elements were prepared. The instrument was optimized as per requirement and results were obtained in ppm levels.

 

Preliminary phytochemical study:

The n-hexane and hydroalcoholic (70% ethanol) extracts were used for phytochemical analysis^22,23,24,25.

 

Determination of total phenol content:

The level of total phenols in hydroalcoholic extract of fruits powder extract was determined by using Folin –Ciocalteu reagent and external calibration with gallic acid^26,27.

 

Determination of flavonoid content

The flavonoid content of hydroalcoholic extract of dried fruit powder extract was determined by aluminium chloride method and quercetin used for external calibration^28,29.

 

Determination of tannin content:

Folin - Ciocalteu method was used for determination of tannins^30,31.

 

Determination of saponin content:

The determination of total saponin was done by standard method³² with some modifications.

 

Determination of alkaloid content:

Hydroalcoholic fruit extract (1mg) was dissolved in dimethyl sulphoxide (DMSO), added 1ml of 2 N HCl and filtered. This solution was transferred to a separating funnel. To it 5ml of bromocresol green solution and 5ml of phosphate buffer were added. The mixture was shaken with 1, 2, 3 and 4ml chloroform by vigorous shaking and collected in a 10ml volumetric flask and diluted to the volume with chloroform. Reference standard solutions of atropine at conc. 20, 40, 60, 80 and 100μg/ml were prepared in the same manner as test solution. The absorbance for test and standard solutions were determined at 470nm against the reagent blank with an UV/Visible spectrophotometer. The total alkaloid content was expressed as mg of AE/g of extract ³³.

 

RESULTS AND DISCUSSION:

Morphological studies:

The color of fresh fruits was green while dried was light brown. Fruits were globose in shape with five cocci and an attached pedicel. Its texture was hard. Odour was slightly aromatic, and taste was slightly bitter and astringent. Surface was ribbed, woody, pubescent and two spines at each coccus showed in figure no.  1.

    

(A) Fresh fruits                        (B) Dried fruits

 

(C)Simple fruit

Fig. 1: fruits of T. terrestris L. (A) color of fresh and dried fruits (B) shape and surface of fruit (C) Simple fruit

 

Microscopic Studies:

Transverse section of fruit and powder study:

Transverse section of fruit showed five cocci filled with fixed oil cells. Epicarp is made up of small tubular cells and containing abundant of unicellular trichomes and fibers. Mesocarp containing vascular bundles and calcium oxalate crystals.  Endocarp consisted of many rows of compactly arranged beaded cells. Endosperm is wide and cells were filled with fixed oil as shown in figure no. 2 (A and B). Powder microscopy of fruits showed numerous trichomes, nonlignified fibers, collenchyma, endosperm cells containing fixed oil, cortex cells, xylem vessels with spiral thickening, sclerenchymatous fibres and sclerenchyma as shown in figure no. 2 (C, D, E, F, G, H, I, J).

Fig. 2  T.S. of fruits of Tribulus terrestris L. (A) five coccus (B) one cocci showing oil cells (O), endopsperm (E), endocarp (ED), mesocarp (M), epicarp (EP), vascular bundles (VB) and trichomes (TR).  Powder microscopy shown by C, D, E, F, G, H, I, J as collenchyma, cortex, non lignified fiber, endosperm cells containing fixed oil, xylem vessels with spiral thickening, glandular trichome, bundle of lignified fibers and sclerenchyma respectively of Tribulus terrestris L. fruits

 

Physicochemical analysis:

The results of various parameters are given in the Table 1. These physicochemical parameters will be functional for identification of Tribulus terrestris fruits even in its powdered form.

 

 

The fruit powder was treated with different chemical reagents and results are reported in Table 2.

 

Table1. Physicochemical parameters of T. terrestris fruits powder

S. No.	Parameter		Value
1	Foreign organic matter		0.6%
2	Loss on drying		14.46±1.85 % w/w
3	Swelling index		54.78± 2.31 % v/v
4	Foaming index		500
5	Volatile oil		Nil
6	Extractive values
	n-hexane extractive value
	Cold maceration method		1.5 % w/w
	Ethanol extractive value
	Hot extraction method		2.76 ±0.22 % w/w
	Cold maceration method		1.38 ±0.27 % w/w
	Aqueous extractive value
	Hot extraction method	5.4 ±0.29% w/w
	Cold maceration method	4.48± 0.2074 % w/w
7	Ash values
	Total ash	10.52±0.60 % w/w
	Acis insoluble ash	1.01±0.06 % w/w
	Water insoluble ash	2.13±0.09 % w/w

Values in % w/w and %v/v are expressed as mean ± SEM; n=3

 

Fluorescence analysis:

Table 2. Fluorescence analysis of powdered T. terrestris fruits

Powdered Drug + reagent	Visible/Day light	UV 254 nm (short)	UV 365 nm (long)
Powder as such	Muddy yellow	Black	Black
Powder + 5% NaOH	Light brown	Black	Black
Powder + acetic acid	Light brown	Black	Black
Powder+distilledwater	Muddy yellow	Black	Dark brown
Powder+conc.H2SO4	Dark brown	Black	Black
Powder+diluteH2SO4	Light brown	Black	Black
Powder + conc. HCl	Brown	Black	Black
Powder+ dil. HCl	Light brown	Black	Dark brown
Powder + ammonia	Pale yellow	Black	Black
Powder+ethylacetate	Light brown	Black	Black
Powder +chloroform	Light brown	Black	Dark brown
Powder + ethanol	Brown	Black	Dark brown

 

Elemental analysis:

The powdered fruits showed elemental contents Pb, As, Co, Zn, Cd, Cu, Hg, Mn, K, Fe and Mg were within limits as showed in the table 3.

 

Table 3: Elemental analysis of T. terristeris L. fruits

Metal	Concentration (ppm)
Lead	1.027
Arsenic	0.000
Copper	0.013
Cadmium	0.008
Mercury	0.000
Magnesium	0.146
Iron	1.559
Manganese	0.022
Calcium	1.305
Potassium	0.740
Cobalt	0.144

 

Preliminary phytochemical screening:

Hydroalcoholic extract of powdered fruits showed the presence of carbohydrates, anthraquinone glycosides, saponins, phenols, tannins, flavonoids, phytosterols while n-hexane extract showed positive results for fatty acid and phytosterols showed in table no. 4.

 

Table 4: Preliminary phytochemical screening of T. territeris fruits

Test	n-hexane extract	70% ethanol extract
Carbohydrate test
Molish test	̶	+
Fehling’s test	̶	̶
Benedict’s test	̶	̶
Glycoside test
Borntrager’s test	̶	̶
Legal’s test	̶	̶
Alkaloid test
Mayer’s test	̶	+
Wagner’s test	̶	+
Dragendroff’s reagent	̶̶	+
Hager’s test	̶	+
Flavonoid test
Alkaline reagent test	̶	+
Lead acetate test	̶	+
H2SO4  test	̶	+
Zinc test	̶	+
Tannin and phenolic compounds
Ferric Chloride test	̶	+
Gelatin – salt Test	̶	+
Iodine Test	̶	+
Nitric acid Test	̶	+
Phytosterol test
Salkowski’s test	+	+
Libermann Burchard’s test	+	+
Saponin test
Froth Test	̶	+
Foam Test	̶	+
Fatty acids test
Spot test	+	+

+= Present, ̶  = absent

 

Total content:

Total phenol, total flavonoid, total tannin, saponin and total alkaloid content is shown in table no. 5. Figure no. 3, 4, 5, 6 showing calibration curve with regression co-efficient (R²).

 

Table 5:  Total phenol, total flavonoid, total tannin, saponin and total alkaloid content of T. territeris fruits

Phytocontent	Value
Total phenol (mg of gallic acid equivalents/g of dry weight)	76.15±1.6
Total flavonoid (mg of quercetin equivalents/g of dry weight)	85.7±0.29
Total tannin (mg of gallic acid equivalents/g of dry weight)	54.1±0.388
Saponin content (mg/g of the dried drug powder)	68±1.73
Total alkaloid (mg of AE/g of dry weight)	35.6 ±0.072

 

 

Fig. 3: Standard calibration curve for total phenolic content

 

 

Fig. 4: Standard calibration curve for total flavonoid content

 

Fig. 5: Standard calibration curve for total tannin content

 

 

Fig. 6: Standard calibration curve for total alkaloid content

 

 

CONCLUSION:

The study was performed to develop the quality control parameters of Tribulus terrestris L. fruits. The results obtained from pharmacognostical studies and phytochemical screening can be used as a diagnostic tool for the standardization of T. terrestris fruits to facilitate quality control and identification of the plant and to minimize the adulteration. Quantitative estimation of secondary metabolites can be used as a potential source of natural bioactive chemicals against pathogenic microorganism and in the screening of the plant drugs for various pharmacological activities.

 

ACKNOWLEDGEMENT:

Authors are thankful to Guru Jambheshwar University of Science and Technology, Hisar for laboratory facility and University Research Scholarship provided to support this research work financially.

 

CONFLICT OF INTEREST:

There is no conflict of interest among authors.

 

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Accepted on 03.05.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5431-5435.