Qualitative and quantitative determination of phytochemicals in Convolvulus leiocalycinus and Haloxylon griffithii

The purpose of the present assessment was to evaluate the phytochemicals in the indigenous plants Convolvulus leiocalycinus of Convolvulaceae family and Haloxylon griffithii of Chenopodiaceae family found in the northern regions of Balochistan. For the analysis of phytochemicals, both plants showed the presence of alkaloids, flavonoids, terpenoids, tannins, saponins, carbohydrates, cardiac glycosides, reducing sugars, quinones and carboxylic acids. Whereas, both plants exhibited negative tests for terpenoids, anthraquinones, phlobatannins, fats, xanthoprotien, resins, anthocyanins, emodins and volatile oil. Furthermore, with the help of standard chemical tests and techniques, the quantitative analysis of C. leiocalycinus and H. griffithii showed different amounts and percentages of total alkaloids, flavonoids, saponins and phenolics. The total alkaloid contents determined were 0.196(W/w) with percentage yield 3.92% and 0.224(W/w) with percentage yield 4.48% in 5gm of each sample of C. leiocalycinus and H. griffithii respectively. The total flavonoid contents determined were 0.28(W/w) with 2.8% and 0.445(W/w) with 4.45% in 10gm of each sample of both the plants. The total saponin contents determined were 0.72(W/w) with 3.6% and 0.53(W/w) with 2.65% in 20gm of each sample of both the plants. The total phenolic contents determined were 0.808(W/w) with 16.16% and 0.268(W/w) with 5.36% in 5gm of each sample of both the plants. The presence of high amount of phytochemical compounds suggest that both plants i.e., C. leiocalycinus and H. griffithii are not only useful to human beings but can also be commercialized for higher production of natural drugs rather than using synthetic drugs with side effects.


Introduction
Phytochemicals are plants based naturally occurring substances.More than ten thousand of these phytochemicals have been identified and many still unknown [1].Phytochemicals include alkaloids, flavonoids, steroids, terpenoids, tannins, phenols, glycosides etc. [2].The therapeutic use of phytochemicals or medicinal plants have been reported in the ancient traditions and cultures of several societies for many centuries.The reason for their wide therapeutic use are low cost and safe nature than synthetic molecules [3,4].Therapeutic processes of phytochemicals involve many biological and pharmacological effects such as antitumoral, anti-microbial, pro-oxidant or anti-oxidant, anti-inflammatory, antiviral and anti-mutagenic activities [5][6][7].Nowadays, positive results in clinical trials have attracted many phytochemicals into medical practices [8].In addition to therapeutic effects, phytochemicals are also used as coloring, flavoring and aromatic agents from time immemorial [9].As stated by World Health Organization (WHO), around 80% people in developing states depend on traditional drugs for their main health requirements and about 85% of these medicines are the plant extracts [10].The fertile land of Pakistan has a wide variety of medicinal plants and many of these need to be explored for their chemical constituents.Such information would be helpful in determining the actual value of folkloric remedies [11].This research study involves for the first time, collection, identification, extraction and phytochemical screening of C. leiocalycinus and H. griffithii.Furthermore, C. leiocalycinus is under shrub having woody branches covered with spines, leaves 12-17 (-24) x (3-) 5-7mm, flowers axillary, solitary, ovary glabrous, style c. 9mm long, c. 3 times the length of the cylindrical stigma.C. leiocalycinus belongs to the family Convolvulaceae.They are annual or perennial shrub found in higher mountainous regions and rugged slopes of Balochistan (Pakistan) [12].On the other hand, H. griffithii is a genus of shrubs or small trees, belonging to the plant family Chenopodiacea, floral leaves reduced on short spikes.Flowers quite distant.Perianth segments ovate, obtuse, till 1.5mm tall.stamens 5; having fruiting perianth wings of white to pinkish or yellowish in color.A very common bush in Balochistan & Khyber Pakhtunkhwa provinces of Pakistan with few records from Chitral and Gilgit.It is also distributed in Afghanistan and Central Asia [13].

Sampling
Different parts of C. leiocalycinus and H. griffithii such as stems, branches, leaves, flowers and roots were collected from different locations of Hanna Lake, Urak region and Spinni road of Quetta, Balochistan on 3 rd of September 2017.The samples were identified by Rasool Bakhsh Tareen, an eminent Botanist, University of Balochistan, Quetta.The samples were dried in shadow, grounded into powder form and sieved (Mesh No 325).The powdered samples were stored in plastic zip bags before use.

Extraction of plant material
The powdered plant substance weighed 4kg was soaked in 15-liter methanol (99.8%) for a period of 7 days.The mixture was filtered by Whatman filter paper No.40 (125mm) followed by re-extraction of the remaining crude with methanol (99.8%) 5 liters unless the color of the solvent changed.Methanol was removed using a rotary evaporator and the dry crude methanolic extract (CME) of C. leiocalycinus having weight 200g and H. griffithii having weight 210g were extracted.The CME of both plants were stored in an airtight container in a cool, dark and dry place and were used for further phytochemical screening.

Phytochemical screening
The plant extracts were further used for the analysis of various phytochemicals qualitatively and quantitatively.

Test for carbohydrates 1. Molisch's test
The plant extract was treated with 10% alcoholic alpha naphthol followed by the addition of 2.5ml sulphuric acid.The presence of carbohydrates and glycosides were confirmed by the appearance of bluish violet region.

Fehling's test
To perform Fehling's test for carbohydrate's presence, the Fehling's solution was prepared and was labeled as A and B. From both the solutions, 5.5ml was added to the plant essence and was heated.The development of reddish brown precipitates was observed for the presence of reducing sugars.

Detection of cardiac glycosides
For cardiac glycosides, 2.5ml of glacial acetic acid was treated with 0.55ml of the plant essence, with the addition of few drops of ferric chloride (5%).To this blend, 1ml of sulphuric acid was mixed and development of a brown ring showed the presence of cardiac glycosides [20].Test for quinones 1.5ml of sulphuric acid was treated with plant extract and a red color formation was noted showing the presence of quinones [21].

Carboxylic acids
For carboxylic acids, 3ml methanolic extract was mixed with 3ml of sodium bicarbonate solution.Effervescence due to carbon dioxide showed the presence of carboxylic acids [22].Quantitative phytochemical analysis Quantitative analysis of the plants extract was conducted to evaluate the amounts and percentages of various phytochemicals such as total alkaloids, flavonoids, phenolics and saponins by means of standard chemical tests and techniques.

Test for total alkaloids
Quantitative estimation of alkaloid was performed by using the procedure of Harborne.Exactly 200cm 3 of 10% acetic acid in ethanol was mixed with each plant powder sample 5gm in a 250cm 3 beaker and kept for 4 hours.The essence was condensed on a water bath to one-fourth of the actual magnitude and then 15 drops of condensed ammonium hydroxide were added drop by drop to the extract.The floating substance was removed, and the precipitates were cleaned through 20cm 3 of 0.1 M ammonium hydroxide solution and then sieved.The filtrate was desiccated in an oven and weighed by electronic balance and the percentage of alkaloid was calculated [23].Test for flavonoids Flavonoids were determined by the procedure described by Bohm and Kocipai-Abyazan.Precisely, 50cm 3 of 80% aqueous methanol was blended to 10gm of sample in a 250cm 3 beaker enveloped and kept for 24 hours at normal temperature.After disposal the floating substance, the remainder extracted thrice with the identical magnitude of ethanol once more.Whatman filter paper having number 42 (125 mm) was utilized to sieve the entire solution of wood specimen.Each specimen remainder was later shifted into a container and desiccated above a water bath.The material in the crucible was cooled through desiccator, weighed and calculated the percentage of flavonoids [24].Test for saponins Saponin quantitative estimation was performed by means of the modern procedure.Accurately, 100cm 3 of 20% aqueous ethanol was mixed with 20 grams of each plant powder sample in a 250cm 3 conical flask.The mixture was heated above a hot water bath for 4 hours with constant stirring at a temperature of almost 55°C.The remainder of the mixture was extracted once more with another 100cm 3 of 20% aqueous ethanol after filtration and heated for 4 hours at a persistent temperature of 55°C with continuous stirring.The collective decoction was vaporized to 40cm 3 above water bath at 90°C.After this, 20cm 3 of diethyl ether was mixed to the distillate in a 250cm 3 separating funnel and strongly blended.Afterward, the aqueous layer was retrieved whereas the ether layer was removed.This refinement procedure was repeated two times.Then, 60cm 3 of nbutanol was mixed and extracted two times with 10cm 3 of 5% sodium chloride.Later removing the sodium chloride layer, the residual solution was heated through water bath for half an hour.After that, the solution was shifted into a container and was desiccated by an oven to get a constant weight.The saponin extract was weighed and computed the percentage [25].

Test for total phenolics
The estimation of total phenolics was accomplished quantitatively using the procedure described by Hagerman.Through this procedure, 5g of powder plant samples were treated separately with 200ml of n-hexane twice for 3 hours each.The filtrate was removed from the residue for the preparation of fat free sample.Then residue was heated on water bath for 20 minutes with 100ml diethyl ether twice and then cooled to room temperature.The solution was filtered and was transferred in a separating funnel.The filtrate was treated with 50ml of 10% NaOH solution two times and was quivered well each time.The organic layer was isolated from the aqueous layer.It was washed two times with 50ml deionized water.The aqueous layer was acidulated up to pH 4 by the addition of 10% HCl solution and 100ml dichloromethane.
Subsequently, the organic layer was collected and dried over water bath, weighed and determined the percentage of phenolics [26].

Results and discussion
The purpose of the current investigation is to assess the phytochemical composition of the methanolic extract of C. leiocalycinus of Convolvulaceae family and H. griffithii of the Chenopodiaceae family by qualitative methods and also to determine the amounts of phytochemicals by quantitative analysis using various standard procedures.Natural products are not only a good source of phytochemicals utilized as drugs but also assist in the manufacture of new effective synthetic drugs.The phytochemical analysis of these indigenous plants showed very exciting results.The screening of the phytochemicals extracted from these plants showed positive tests for the presence of alkaloids, flavonoids, tannins, phenolics, saponins, carbohydrates, glycosides, quinones and carboxylic acids given in the (Table 1).However, phytochemicals such as terpenoids, anthraquinones, phlobatannins, fats, xanthoprotien, resins, emodins, volatile oil, and anthocyanins showed negative tests for both plants given in the (Table 2).

Quantitative Analysis
The quantitative estimation of the examined plants revealed that phytochemicals are present in different amounts in each plant extract.The total alkaloid contents determined were 0.196(W/w) with percentage yield 3.92% and 0.224(W/w) with percentage yield 4.48% in 5gm of each sample of the C. leiocalycinus and H. griffithii.The result showed that alkaloid content was greater in H. griffithii than C. leiocalycinus (Table 3).Similarly, the total flavonoid contents determined were 0.28(W/w) with percentage yield 2.8% and 0.445(W/w) with percentage yield 4.45% in 10gm of each sample of both the plants.The flavonoid content was higher in H. griffithii than C. leiocalycinus (Table 4).Moreover, the total saponin contents determined were 0.72(W/w) with percentage yield 3.6% and 0.53(W/w) with percentage yield 2.65% in 20gm of each sample of both the plants.The results revealed that the concentration of saponins was higher in C. leiocalycinus as compared to H. griffithii (Table 5).Consequently, the total phenolic contents determined were 0.808(W/w) with percentage yield 16.16% and 0.268(W/w) with percentage yield 5.36% in 5gm of each sample of C. leiocalycinus and H. griffithii.
The phenolic contents were higher in C. leiocalycinus than H. griffithii (Table 6).Concluding these results, it can be simply judged that the phenolic contents are higher than the contents of alkaloids, flavonoids and saponins of both the plants.The results from the quantitative analysis showed significant variations among the contents of alkaloids, flavonoids, saponins and phenolics when compared with one other.