Comprehensive Phytochemical Screening and Quantification of Bioactive Compounds in Ziziphus spina-christi for Herbal Cosmetics

Shaziya Yasmeen Sayeed*; Anju Goyal

Bhupal Nobles’ College of Pharmacy, Udaipur (India)

*Correspondence: shaziya.sy@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2026.5106

INTRODUCTION

Ziziphus spina-christi, commonly known as “Sidr” or “Christ’s Thorn,” is an evergreen shrub or small tree of the Rhamnaceae family, native to the arid and semi-arid regions of Africa, the Middle East, and South Asia. It holds a significant place in traditional medicine systems, especially for hair and scalp care, due to its cleansing, antimicrobial, and anti-inflammatory properties. The leaves, in particular, have been used for centuries as a natural shampoo and conditioner in many cultures, notably in the Middle East.(1,2)

Recent phytochemical investigations have revealed that the leaves of Ziziphus spina-christi contain a rich variety of bioactive compounds, including saponins, flavonoids, tannins, alkaloids, triterpenoids, and phenolic acids. Saponins act as natural surfactants conferring cleansing ability, while flavonoids and phenolics provide potent antioxidant effects through free radical scavenging activity, which can protect hair follicles and scalp skin from oxidative damage. Additionally, triterpenes and other phytochemicals present in the plant exhibit anti-inflammatory and antimicrobial activities, further supporting its traditional uses and potential incorporation into herbal cosmetic formulations.(1,3)

The increasing consumer preference for herbal and sustainable personal care products has amplified research interest in Ziziphus spina-christi to scientifically validate its functional benefits and safety. This study aims to evaluate the phytoconstituents and antioxidant (free radical scavenging) activity of Ziziphus spina-christi leaf extract with the goal of formulating an effective and safe herbal shampoo, thereby bridging traditional knowledge with modern phytochemical and cosmetic science.(4–6)

Materials and Methods

Plant Material

Fresh leaves of Ziziphus spina-christi were collected, thoroughly washed, shade-dried, and pulverized to fine powder.

Preparation of Extract

Extract  was  prepared  from  fresh  leaves  of Z. spina-christi using Soxhlet extractor apparatus. 15 grams of fresh leaves were placed in the Soxhlet extractor and then 250 ml of 70% ethanol were added to the distillation flask. After refluxing  for  8  hr,  the  solvent  was  evaporated  under  reduced pressure. (30:70 water and ethanol). (7)

Phytochemical profiling of Ziziphus spina-christi

Qualitative tests were performed for major phytochemical groups:

Tests For Carbohydrates

The presence of carbohydrates was investigated using several qualitative assays:

Molisch test: To 1 ml of the extract, 2-3 drops of alcoholic α-naphthol solution were added. Concentrated sulphuric acid was then gently introduced along the side of the test tube. The formation of a purple ring at the interface between the two liquids indicated the presence of sugars.

Fehling’s test: Equal volumes (1 ml each) of Fehling’s solutions A and B were combined with 1 ml of the extract and heated in a water bath for several minutes. The appearance of a brick-red precipitate confirmed the presence of carbohydrates.

Benedict’s test: Equal parts of Benedict’s reagent and the extract were mixed and boiled for 5 to 10 minutes. A colour shift from green to yellow or red, depending on the concentration of reducing sugars, indicated their presence.

Barfoed’s test: A few drops of Barfoed’s reagent were added to 1 ml of the extract followed by heating in a water bath for 2 minutes. The development of a red coloration, due to cupric oxide formation, revealed the presence of monosaccharides. (8-9)

Tests for Alkaloids

Alkaloid testing was carried out using the following standard assays:

Dragendorff’s test: Addition of Dragendorff’s reagent (potassium bismuth iodide) to the drug solution resulted in the formation of an orange-red coloration, confirming alkaloids.

Mayer’s test: Upon adding Mayer’s reagent (potassium mercuric iodide), a creamy white precipitate developed, indicative of alkaloids.

Hager’s test: The addition of Hager’s reagent (a saturated aqueous solution of picric acid) yielded a yellow crystalline precipitate in the presence of alkaloids.

Wagner’s test: When Wagner’s reagent (dilute iodine solution) was added, a reddish-brown precipitate formed, confirming alkaloids.

Tannic acid test: A buff-colored precipitate was observed upon the addition of tannic acid to the drug solution, further confirming alkaloids. (10,11)

Tests for Glycosides

Anthraquinone Glycosides

Borntrager’s test: One gram of the drug was boiled with 5–10 ml of dilute hydrochloric acid for 10 minutes and then filtered. The filtrate was extracted with carbon tetrachloride or benzene, followed by addition of an equal volume of ammonia. The formation of a pink or red coloration in the ammoniacal layer signified the presence of anthraquinone glycosides.

Modified Borntrager’s test: One gram of the drug was treated with dilute hydrochloric acid and 5% ferric chloride, boiled for 10 minutes, cooled, and filtered. The filtrate was extracted with carbon tetrachloride or benzene, and upon addition of ammonia, a pink to red coloration developed, confirming C-type anthraquinone glycosides. (12)

Saponin Glycosides

Foam test: When 1 g of the extract was shaken with 10–20 ml of water, persistent frothing for 60 to 120 seconds was indicative of saponins.

Froth test: Extract was diluted with distilled water to 20ml and this was shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins. (13)

Steroid and Triterpenoid Glycosides

Libermann-Burchard test: The alcoholic extract was evaporated to dryness, extracted with chloroform, and then treated with acetic anhydride followed by concentrated sulfuric acid. The appearance of a violet to blue ring at the junction of the two layers confirmed the presence of steroids.

Salkowski test: Following chloroform extraction of the evaporated alcoholic extract, addition of concentrated sulfuric acid resulted in a yellow ring that turned red after two minutes, indicative of steroids.

Cardiac Glycosides

Keller-Kiliani test: The alcoholic extract was mixed with water and lead acetate, shaken, and filtered. The filtrate was extracted with chloroform, evaporated to dryness, and the residue dissolved in glacial acetic acid. Addition of ferric chloride followed by concentrated sulfuric acid resulted in a reddish-brown layer turning bluish-green on standing, confirming digitoxose.

Legal test: The alcoholic extract was treated with water, lead acetate, and chloroform as above. The dried residue was dissolved in pyridine, mixed with sodium nitroprusside, and made alkaline to produce a pink color, indicating glycosides.

Baljet test: Dipping leaf sections or drug parts containing cardiac glycosides in sodium picrate solution yielded a yellow to orange color, indicative of aglycones or glycosides. (14)

Flavonoid Glycosides

Ammonia test: Filter paper impregnated with the alcoholic extract showed a yellow spot upon exposure to ammonia vapor.

Shinoda test: Addition of magnesium turnings and dilute hydrochloric acid to the alcoholic extract produced a red color suggestive of flavonoids; substitution with zinc turnings yielded a deep red to magenta color indicative of dihydroflavonoids.

Vanillin-HCl test: Addition of vanillin-HCl produced a pink coloration, supporting the presence of flavonoids. (15)

Tests for Tannins and Phenolics

Vanillin-hydrochloric acid test: Treatment with a mixture of vanillin, alcohol, and dilute hydrochloric acid generated a pink or red coloration, linked to phloroglucinol formation.

Ferric chloride test: Addition of alcoholic ferric chloride to concentrated extract caused a deep green color that changed to yellow with concentrated nitric acid, indicating coumarins.

Gelatin test: Mixing the extract with 1% gelatin solution resulted in a buff-colored precipitate, confirming tannins.

Lead acetate test: The presence of tannins was further confirmed by the appearance of a precipitate upon addition of lead acetate. (16)

Quantitative phytochemical analysis

a)      Total saponin content

Saponins are the primary surfactants in Ziziphus spina-christi. They contribute to foaming, emulsification, cleansing, and detergent activity in shampoo.

To make a standard saponin solution, 110 mg of extract was dissolved in 16 milliliters of methanol and 4 milliliters of purified water. This was then further diluted to create various concentrations (100–600 µg/ml) to estimate the total saponin content. Sulphuric acid (72% v/v, 2.5 ml) and vanillin reagent (8%, 0.25 ml) were gradually added to each concentration. After thoroughly combining the solutions, the tubes were placed in a water bath set at 60°C. After incubating for ten minutes, the tubes were cooled in an ice-cold water bath for three to four minutes. The absorbance was measured at 544 nm with respect to the reagent blank. The same procedure as the standard was followed to prepare the samples, and absorbance was recorded. All the experiments were carried out in triplicate. Total saponin content is calculated by using the formula: C = cV/m, where C = total saponin content mg DE/g dry extract, c = concentration of diosgenin obtained from the calibration curve in mg/ml, V = volume of extract in ml, and m = mass of extract in grams. (17)

b)      Total Phenolic Content (TPC)

Phenolics provide antioxidant activity, help prevent scalp irritation, and protect against oxidative damage to hair and scalp.

Gallic acid solutions in methanol were prepared in different concentrations (10, 20, 30, 40, 50, and 60 µg/ml). 1 ml of gallic acid of each concentration was added in different test tubes, and 5 ml of Folin-Ciocalteu reagent (10%) and 4 ml of 7% Na₂CO₃ were added to get a total volume of 10 ml. The blue-colored mixture was shaken well and incubated for 30 minutes at 40° C in a water bath. Then, the absorbance was measured at 760 nm against the blank. Every experiment was run in triplicate. The calibration curve was plotted using the average absorbance values obtained at various gallic acid concentrations.

The milligrams of gallic acid equivalents (GAE) per gram of dry extract (mg/g) were used to report the extracts' total phenolic content.

The equation C = cV/m was used to determine the entire phenolic content in every sample. Here, C stands for the entire phenolic compound in mg of gallic acid equivalent/g dry extract, c for gallic acid concentration measured in mg/ml from the curve used for calibration, V for extract volume in milliliters, and m for extract mass

in grams. (18)

c)       Total Flavonoid Content (TFC)

Flavonoids improve scalp microcirculation, have anti-inflammatory and antimicrobial properties, and contribute to hair health.

Standard quercetin was prepared in methanol at different concentrations (20, 40, 60, 80, and 100 µg/ml). An aliquot of 1 ml of quercetin of each concentration was added to a 10 ml volumetric flask containing 4 ml of distilled water. 0.3 ml of 5% sodium nitrite was added at zero time, followed by 0.3 ml of 10% AlCl₃ and 2 ml of 1 M sodium hydroxide at six minutes. Distilled water was then added to bring the mixture's total volume to 10 ml, and the mixture turned pink. The mixture's absorbance was measured at 510 nm against a blank that contained all of the reagents except quercetin. The

calibration curve was plotted using the average absorbance values obtained at various quercetin concentrations.

Samples were prepared, and the same methodology was used to measure absorbance as the standard. The extracts' overall flavonoid content was reported in mg of equivalents of quercetin (QE) in one gram of dry extract (mg/g). Every experiment was run in triplicate. C = cV/m  is the formula used to calculate total flavonoid content. Here, C stands for total flavonoid content mg QE/g dry extract, c is the quercetin concentration measured in mg/ml from the calibration curve, V is the extract volume in milliliters, and m is the extract mass in grams. (19,20)

Results

Qualitative phytochemical analysis:

Qualitative phytochemical analysis demonstrated positive presence (+) of carbohydrates (Molisch’s, Fehling’s, Benedict’s, Barfoed’s tests), alkaloids (Dragendorff’s, Mayer’s, Wagner’s, Hager’s, tannic acid tests), saponins (foam and froth tests), steroids and triterpenoids (Libermann–Burchard and Salkowski tests), cardiac glycosides (Keller–Kiliani, Legal, Baljet tests), flavonoids (Shinoda, ammonia, vanillin-HCl tests), and tannins and phenolics (ferric chloride, gelatin, lead acetate, vanillin-HCl tests). Anthraquinone glycosides were absent as indicated by negative Borntrager’s and modified Borntrager’s tests.

Quantitative phytochemical analysis:

a). Total Saponin Content (TSC)

Table: Absorbance of Diosgenin

Figure:

b). Total Phenolic Content (TPC)

Table: Absorbance of Gallic Acid

Figure:

c). Total Flavonoid Content (TFC)

Table: Absorbance of Quercetin

Figure:

These results indicate a high concentration of saponins, phenolics, and flavonoids, which are responsible for the extract’s antioxidant capacity, anti-inflammatory potential, and surfactant properties relevant to shampoo formulation.

Conclusion

The comprehensive phytochemical screening and quantification confirm that Ziziphus spina-christi leaf extract is rich in bioactive compounds such as saponins, phenolics, and flavonoids, which underpin its traditional use in hair and scalp care. The significant antioxidant activity and presence of natural surfactants support its efficacy and safety as an ingredient in herbal shampoo formulations. This study scientifically validates the ethnomedicinal applications of Sidr and provides a phytochemical basis for its development into effective and sustainable herbal cosmetic products.

References

1.             Batovska D, Gerasimova A, Nikolova K. Exploring the Therapeutic Potential of Jujube (Ziziphus jujuba Mill.) Extracts in Cosmetics: A Review of Bioactive Properties for Skin and Hair Wellness. Cosmetics. 2024 Oct 15;11(5):181.

2.             Asgarpanah J. Phytochemistry and pharmacologic properties of Ziziphus spina christi (L.) Willd. Afr J Pharm Pharmacol. 2012 Aug 22;6(31).

3.             El-Shahir AA, El-Wakil DA, Abdel Latef AAH, Youssef NH. Bioactive Compounds and Antifungal Activity of Leaves and Fruits Methanolic Extracts of Ziziphus spina-christi L. Plants. 2022 Mar 11;11(6):746.

4.             Elghaffar RYA, Amin BH, Hashem AH, Sehim AE. Promising Endophytic Alternaria alternata from Leaves of Ziziphus spina-christi: Phytochemical Analyses, Antimicrobial and Antioxidant Activities. Appl Biochem Biotechnol. 2022 May 17;194(9):3984–4001.

5.             Hussein NN, Al-Azawi K, Sulaiman GM, Albukhaty S, Al-Majeed RM, Jabir M, et al. Silver-cored Ziziphus spina-christi extract-loaded antimicrobial nanosuspension: overcoming multidrug resistance. Nanomedicine (Lond). 2023 Oct 1;18(25):1839–1854

6.             Shnawa BH, Jalil PJ, Al-Ezzi A, Mhamedsharif RM, Mohammed DA, Biro DM, et al. Evaluation of antimicrobial and antioxidant activity of zinc oxide nanoparticles biosynthesized with Ziziphus spina-christi leaf extracts. Journal of Environmental Science and Health, Part C. 2023 Dec 11;ahead-of-print(ahead-of-print):93–108.

7.             Ali HS, Kadhim RB. Formulation and evaluation of herbal shampoo from Ziziphus spina leaves extract. Int J Res Ayurveda Pharm. 2011;2(6):1802–1806.

8.             Khandelwal KR. Practical pharmacognosy: techniques and experiments. 29th ed. Pune: Nirali Prakashan; 2021. p. 149-52.

9.             Trease GE, Evans WC. Pharmacognosy. 16th ed. London: Saunders; 2009. p. 45-7.

10.         Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 55th ed. Pune: Nirali Prakashan; 2021. p. 225-8.

11.         Khandelwal KR. Practical pharmacognosy: techniques and experiments. 30th ed. Pune: Nirali Prakashan; 2023. p. 157-60.

12.         Harborne JB. Phytochemical methods: a guide to modern techniques of plant analysis. 3rd ed. London: Chapman & Hall; 1998. p. 170-2.

13.         Evans WC. Trease and Evans’ pharmacognosy. 16th ed. Edinburgh: Saunders Elsevier; 2009. p. 247–249.

14.         Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 50th ed. Pune: Nirali Prakashan; 2014. p. 7.19–7.24, 7.31–7.35.

15.         Krishnaiah, D., Devi, T., Bono, A., & Sarbatly, R. (2009). Studies on phytochemical constituents of six Malaysian medicinal plants. Journal of Medicinal Plants Research, 3, 67-72.

16.         Patel, R., Patel, A., Desai, S., & Nagee, A. (2012). Study of secondary metabolites and antioxidant properties of leaves, stem, and root among Hibiscus rosa-sinensis cultivars. Asian Journal of Experimental Biological Sciences, 3(4), 719-725.

17.         Mohaddes-Kamranshahi M, Jafarizadeh H, Simjoo M, Jafarizad A. Evaluation of the saponin green extraction from Ziziphus spina-christi leaves using hydrothermal, microwave and Bain-Marie water bath heating methods. Green Process Synth. 2018;8. doi:10.1515/gps-2017-0185.

18.         Singleton VL, Orthofer R, Lamuela-Raventós RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods Enzymol. 1999;299:152-78.

19.         Chang CC, Yang MH, Wen HM, Chern JC. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal. 2002;10(3):178-82.

20.         Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 1999;64(4):555–559.