BETA AND GAMMA SITOSTEROL; AN ENVIRONMENTAL

EXPLORATION FROM D. PINNATA LEAVES FOR DEMOCRAT’S

WELLNESS

Yakubu Rufai*1, 2, Omar Abdullahi Wafudu Handy4, Sani Aliyu1,3, Muhammad Isah Bello2
1
Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia (UTM), 81310 Johor
Bahru, Johor, MALAYSIA
2
Chemistry Department, Federal College of Education (FCE) Okene P.M.B 1062 Kogi State, NIGERIA
3
Department of Chemistry, Kaduna State University, P.M.B 2339, Tafawa Balewa Way, Kaduna, Nigeria
4
Federal Polytechnic Mubi, SLT Department Adamawa State-Nigeria
*
Corresponding authors email address: yakuburufaibaby@gmail.com

Abstract
The role of environmental exploration especially plants natural sources and the democrat’s
useability in facilitating effective governance is less emphasized. They are not only used as food,
Inspiration, income and shelter but contribute to human health/wellbeing. Phytochemical
analysis of Deinbollia pinnata leave extracts exhibited the presence of cardiac glycosides,
coumarins, reducing sugars, steroids, flavonoids, tannins, and phenolic compounds. βeta-
sitosterol (1), and Gamma-sitosterol (2) were isolated for the first time from the leaf’s ethyl
acetate extracts by chromatographic methods. Their structures were elucidated based on
spectroscopic analysis such as infrared (IR), gas chromatography-mass spectroscopy (GC-MS),
nuclear magnetic resonance (NMR), and comparison with data from the literature. The
preclinical evidence for both compounds from potential clinical applications reported their
abundantly uses, which active democrats shall depend on its use for physiological and
phycological anti-stress.

INTRODUCTION

Governance through democratic rules seems to be accepted throughout the world (Wike et al.,
2017). Its political systems are concretely structured to generate legitimate laws promoting
effective inclusive, equitable economics, sustainability and social progress through fair elected
transparent voting. It stands to govern well today, emerging issues and future demands of the
society (Amanitidou, 2016). Fortunately, challenges of modern societies' dominant characters
with complexity, uncertainty and diversity have put the democrats in serious physiological and
phycological tremors leading to serious illness. Thus, the medical practitioners' efforts in the
management of common diseases especially lowering cholesterol levels and improving

1
symptoms of an enlarged prostate still pose a significant threat to humanity globally which are
commonly found among the democrats due to stressful activities they undergo. More so, the
associated side effects of conventional drugs for disease treatment outrage the intended treated
illness (Zumthurm, 2020). The present poverty level has cut off the purchase of medicine even at
the point of death. It is, therefore necessary to search for natural sources for effective, safe, and
affordable medicines for human wellbeing. Plant parts were believed to contain phytochemicals
used in the management of several diseases from time immemorial. Today, they are researched
effectively to reduce the risk of cardiovascular disease, cancer, Alzheimer's disease, stroke,
cataracts, and help delay ageing (Kasote et al., 2015).

Sitosterol is an important plant sterol which helps to reduce benign prostatic and duodenal ulcer
protection (Rakel, 2018; Tovey, 2018), is used as hair supplements and inhibits rheumatoid
synovial angiogenesis (Nguyen et al., 2019; Qian et al., 2022), cardiovascular disease
management and impedes high cholesterol (Genser et al., 2012; Shi et al., 2011). Also, γ-
sitosterol act as antidiabetic and as anti-cancer (Sundarraj et al., 2012; Tripathi et al., 2013). The
roots act as an antibacterial agent (Sotubo et al., 2016) and leaves are used as a remedy for
several diseases such as febrifuge, analgesic, bronchitis intercostals, intestinal pains, jaundice,
cough, asthma, and infections (Sotubo et al., 2016). Also, leaves decoction is used in positioning
the fetus during childbirth (Kankara et al., 2015). However, despite promising medicinal values
within the phytochemicals available in the literature. This chemical characterization will lead to a
promising source of biologically potent compounds to improve the well-being of the democrats
and non-democrats towards achieving good governance in the country. Therefore, the study was
carried out to evaluate the classes of phytochemicals present in the leaves and further isolate its
compound and how their medicinal uses protect/cure illnesses in humans. To our knowledge, this
is the first report on the isolation of these isomeric sitosterol compounds from D. pinnata leaves.

Materials and Methods

Chemicals and reagents; All chemicals and reagents used in this work were of analytical grade.

Plant Materials

2
The D. pinnata (Poir.) Schumach. & Thonn. Leaves were collected during the dry season,
January 2017 for one week (daytime tempt. ranges from 28°C (82°F) in January to 31/32°C
(88/90°F); the flowering period from Okehi Local Government Area of Kogi State, Nigeria. The
plant parts were identified and confirmed at Biological Department, Federal College of
Education Okene Kogi State by Mrs Aniama S.O.A. a botanist. The plant material was
authenticated at the Forestry Research Institute of Nigeria Ibadan through comparison with the
voucher specimen under accession number FHI 3251 by Mr Michael. The plant samples were
collected, washed and air dried at room temperature for one month and turned to powder as
shown in Figure 1.

Figure 1: (a) Leaves Powder (b) The leaves

The scientific Classification of D. pinnata is as follows:

Family: Sapindaceae
Genus: Deinbollia
Species: pinnata
Common name: water willow
Yoruba: Ogiri-egba
Egbo: Ekusi-Oloko
Egbirra: Ereto

General Experimental Procedure

1
H and C NMR spectra were recorded on Bruker Avance (400 MHz) spectrometer, using
13

deuterated chloroform (CDCl3) as the solvent. The IR was recorded on a PerkinElmer FT-IR

3
spectrometer, while GC-MS data were acquired using Agilent 7820A (G4350). Thin-layer
chromatography (TLC) was done on 0.20 mm precoated silica gel aluminium sheets (Merk
Kieselgel 60 F254). TLC spots were observed under the UV light at 254 nm and 365 nm.
Vanillin sulphuric acid was used as the spraying reagent. Extraction and phytochemical
screening. The D. pinnata (Poir.) Schumach. & Thonn. leaves were air-dried and triturated to a
coarse powder using a grinding machine. Each sample (50 g) was separately macerated with
methanol, and another set (50 g each) was extracted using Soxhlet for comparison of yield and
phytochemicals present. The extracts were concentrated under a vacuum using a rotary
evaporator to obtain D. pinnata (Poir.) Schumach. & Thonn. leaves extracts from maceration and
Soxhlet methods.

Phytochemical screening of the D. pinnata (Poir.) Schumach. & Thonn. leaves

Preliminary phytochemical screening of D. pinnata (Poir.) Schumach. & Thonn. leaves were
conducted using the standard procedures with slight modifications as follows;

Tests for simple sugar (Fehling’s test)

Approximately 1 mL of Fehling’s solutions A and B were mixed and warmed for 1 minute. The
mixture was added to an equal volume of the test solution. The solution was heated in a boiling
water bath for 5-10 minutes. A yellow precipitate was formed, which then turned to brick red.

Test for anthraquinone glycosides (Borntrager’s test)

Each test sample was mixed with 3 mL of dilute H2SO4, warmed, and filtered. The filtrate was
cooled, and an equal volume of benzene was added to it. The solution was shaken vigorously to
separate the organic layer. An equal volume of dilute ammonia solution was added to the organic
layer. The inability of the ammonia layer to turn pink indicates the absence of anthraquinone
glycosides.

Test for cardiac glycosides (Keller-Killiani test)

A volume of 1 mL concentrated H2SO4, 2 mL of glacial acetic acid, and 1 drop of FeCl3 solutions
was added to 5 mL of the extract. The appearance of a brown ring indicated the presence of
cardiac glycosides.

Test for coumarins

4
The concentration of 10% NaOH was added to 2 mL of the extract. The mixture was shaken
vigorously for 5 min. The appearance of yellow colouration indicated the presence of coumarins.

Test for saponins (foam test)

About 20 mL of distilled water was added to 1 mL of the extract in a measuring cylinder. The
mixture was shaken well for 15 min, and no layer of foam was observed.

Tests for quinone

Concentrated H2SO4 was mixed with 2 mL of the extract in a test tube and shaken vigorously for
5 min. No visible red colour was observed.

Test for steroids (Salkowski test)

About 2 mL of concentrated H2SO4 and 2 mL of chloroform were added to 2 mL of the extract.

The solution was shaken vigorously. The chloroform layer changed to red, while the acid layer
showed greenish-yellow fluorescence.

Test for alkaloids (Mayer’s test)

Approximately 1 mL of dilute HCl and 1 mL of Mayer’s reagent were added to 2-3 mL of

filtrate, and well shaken. The formation of yellow precipitate indicated the presence of alkaloids.

Test for Flavonoids (Shinoda test)

Concentrated hydrochloric acid (HCl) was added in drops to the test solution. Magnesium
turnings were put into the solution. The pink-red colour observed indicated the presence of
flavonoids.

Test for tannins and phenolic

Compounds FeCl3 solution test A solution of 5% FeCl 3 was added to 2 mL of the extract. A deep
blue-black colouration indicated tannins and phenolic compounds in the test sample.

Extraction and isolation procedure

5
The powdered D. pinnata (Poir.) Schumach. & Thonn. leaves (1.5 kg) were macerated
successively in increasing solvent polarity with n-hexane, ethyl acetate, and methanol each for
72 hours. The extracts were separately concentrated under vacuum yielding n-hexane, ethyl
acetate, and methanol crude extractives. Silica gel 60 Å (Mesh size 230–400) was used for
Vacum Liquid Chromatography (VLC) of the EtOAc extracts (extract: silica gel = 1:30) yielding
35 fractions. Fractions were pooled based on TLC profiles resulting in five sub-fractions and
were labelled as A–E. Sub-fraction B (3.48 g) was further fractionated using VLC and 44 sub-
fractions were obtained, which were then pooled based on the Thin-Layer Chromatography
(TLC) profile. Further purification of DPL2 by column chromatography (CC) resulted in the
isolation of isomeric sitosterol; beta and gamma sitosterol.

Results and Discussions

The yield of extraction of D. pinnata (Poir.) Schumach. & Thonn. leaves using the maceration
method are shown in Table 1 with various physical appearances. Methanol extracts yielded
higher (73.92 g, 4.93%) and ethyl acetate with a lower yield of (20.91 g, 1.39%). N- hexane was
in moderate yield. The results of phytochemical screening of D. pinnata (Poir.) Schumach. &
Thonn extracts revealed the presence of phenolics, reducing sugars, saponins, coumarins,
steroids, flavonoids, tannins, and alkaloids as shown in Table 2. The deep colouration appeared
which showed a high presence of phytochemicals found for alkaloids, phenols, steroids and
reducing sugar. Therefore, the plant under study may possess bioactive compounds with specific
medicinal values.

Table 1: Yield and Physical Appearance of Deinbollia pinnata (Poir.) Schumach. & Thonn.
Leave Extracts

Solvent (s) Leaves (g, %) Physical appearance

n-Hexane (48.25 g, 3.22%) Brown, gummy
Ethyl acetate (20.91 g, 1.39%) Brown, gummy
Methanol (73.92 g, 4.93%) Dark red, gummy
1.5 kg leaves were used in the experiment performed; values are expressed in both grams/percentages .

Table 2: Phytochemical Analysis for D. pinnata (Poir.) Schumach. & Thonn. leave extracts

6
 Constituents Test Reagent/Chemical Leaf
Alkaloids Hager’s reagent ++
Reducing sugar Fehling’s reagent +++
Steroids Salkowski ++
Saponins Foam +
Tannins Lead acetate sol. +
Phenolics FeCl3 solution +++
Fixed oil & fats Spot test ‒
Protein Biuret’s reagent ‒
Anthraquinone Borntrager’s reagent ‒
Cardiac glycosides Kelle-killiani’s reagent ‒
Lead acetate +
Flavonoids
Extract +NH3(aq.) +
Quinones Extract + Conc. H2SO4(aq) ‒
Coumarins Extract +10% NaOH(aq.) ++

Isolation and purification of compounds from D. pinnata (Poir.) Schumach. & Thonn ethyl
acetate extracts using gravity column chromatographic separation on DPEL2 yielded the two
compounds. γ- Sitosterol was isolated as white solid (13.0 mg) Rf = 0.55 in PE: EtOAc 4:1.
Treatment of the TLC spot with vanillin sulphuric acid reagent gave a purple colouration,
indicating a terpene type of compound. The IR spectrum of (1)) shows a broad absorption band
at 3390 cm-1 characteristics of the O-H group, while the band at 2933 cm-3 corresponds to the
sp3 C-H stretch. The 1H NMR of compound (1)) shows chemical shifts at δ 0.69 (3H, s, H- 18), δ
1.02 (3H, s, H-19), (3H, d, J=6.4 Hz, H-21), δ 0.82 (3H, d, J=2.0 Hz, H-26), δ 0.84 (3H, d, J=2.0
Hz, H-27), δ 0.86 (3H, H-29) representing methyl groups in the compound. Evidence for oxy-
methine proton at position C-3 was indicated by the peak at δ 3.54. The signal at δ 5.37 was for
the proton attached to C=C at position C-6. 13C chemical shifts at δ 121 and δ 140 confirm the
presence of olefinic carbons C-6 and C5, respectively. Oxymethine carbon C-3 was represented
by the signal at δ71.82. The various peaks at δ 11.85–19.80 represent the methyl groups. The

7
GC-MS spectrum showed a molecular ion peak [M+] at m/z 414.5 (calc. 414) corresponding to
the molecular formula of C29H50O (Yakubu et al., 2014).

Figure 2: 3d structure of beta-sitosterol 3d structure of gamma sitosterol

Compound (2) was obtained as colourless needles (10 mg, 0.33%), with m.p. 145°C (Pierre &
Moses, 2015) 147-148°C). Their TLC profiles gave a purple spot after spraying with vanillin
sulphuric acid reagent which suggested a terpene-type of compounds. Compound (2) showed an
absorption band of sp3 for C-H stretching at 2935 cm-1, a typical hydroxyl group absorption band
at 3406 cm-1, and C-O stretching at 1052 cm-1. Compound (2) revealed a molecular ion, M+ at
m/z 412 corresponding to the molecular formula C29H48O. Based on the earlier report of 1H and
13
C NMR data (Bulama et al., 2015).

Spectroscopic Data for Isolated Compounds

γ- Sitosterol (1) was obtained from the purification of n-hexane extract as colourless needles. (13
mg); m.p. 146-148°C; Rf 0.65; IR (Neat) vmax cm-1 (CDCl3): 3390 (OH), 2933 (C-H) and 1051
(C-O); 1H-NMR (CDCl3, 400 MHz): δH 3.50 (1H, m, H-3), 5.36 (1H, t, J = 2.16 Hz, H-6), 0.69
(3H, s, H- 18), 0.93 (3H, d, J = 6.4 Hz, H-21), 0.82 (3H, d, J = 2.0 Hz, H-26), 0.84 (3H, d, J = 2.0
Hz, H-27), 0.86 (3H, H-29). 13C-NMR (CDCl3, 100 MHz): δC 37.26 (C-1), 32.41 (C-2), 71.82
(C-3), 42.26 (C-4), 140 (C-5), 121.71 (C6), 31.92 (C-7), 31.63 (C-8), 50.23 (C-9), 33.72 (C-10),
21.09 (C-11), 39.79 (C-12), 42.33 (C-13), 56.08 (C14), 26.13 (C-15), 28.24 (C-16), 56.78 (C-
17), 11.85 (C18), 19.39 (C-19), 36.51 (C-20), 19.04 (C-21), 33.97 (C-22), 26.13 (C-23), 45.86
(C-24), 29.19 (C-25), 18.25 (C-26), 19.80 (C-27), 23.09 (C-28), 12.22 (C-29). GC-MS: 57, 69,

8
81, 95, 105, 119, 133, 145, 159, 173, 187, 199, 213, 222, 231, 241, 255, 273, 283, 303, 329, 341,
354, 371, 381, 396, 414. EIMS; m/z: 414 [M]+ (C29H50O).

β-sitosterol (2): was obtained as colourless needles (10 mg), with m.p. 145°C (Pierre & Moses,
2015) 147-148°C).; Rf 0.65; IR (Neat) νmax cm-1; 2933 (C-H), 3391 (OH), 1051 (C-O); 1H NMR
(400 MHz, CDCl3): δH 1.25 (2H, m, H-1), 1.47 (2H, m, H-2), 3.51 (1H, m, H-3), 2.00 (2H, m, H-
4), 5.35 (1H, t, J = 2.16 Hz, H-6), 1.85 (2H, m, H-7), 1.45 (1H, m, H-8), 1.44 (1H, m, H-9), 1.42
(2H, m, H-11), 1.35 (2H, m, H-12), 1.42 (1H, m, H-14), 1.47 (2H, m, H-15), 1.48 (2H, m, H-16),
1.47 (1H, m, H-17), 0.69 (3H, s, H-18), 1.02 (3H, s, H-19), 1.53 (1H, m, H-20), 0.93 (3H, d, J =
6.4 Hz, H-21), 1.25 (2H, m, H-22), 1.25 (2H, m, H-23), 0.98 (1H, m, H-24). 1.86 ((2H, m, H-25),
0.82 (3H, t, J = 6.0 Hz, H-26), 0.83 (1H, m, H-27), 1.29 (3H, d, J = 8.0 Hz, H-28), 0.85 (3H, d, J
= 7.6 Hz, H-29); 13C NMR (400 MHz, CDCl3); 37.2 (C-1), 32.4 (C-2), 71.8 (C-3), 42.2 (C-4),
140.7 (C-5), 121.7 (C-6), 31.9 (C-7), 31.6 (C-8), 50.2 (C-9), 33.7 (C-10), 21.0 (C-11), 39.7 (C-
12), 42.3 (C-13), 56.0 (C-14), 26.1 (C-15), 28.2 (C-16), 56.7 (C-17), 11.8 (C-18), 19.3 (C-19),
36.5 (C-20), 33.9, 19.0 (C-21), (C-22), 26.1 (C-23), 45.8 (C-24), 29.1 (C-25), 18.2 (C-26), 19.8
(C-27), 23.0 (C-28), 12.2 (C-29). EIMS; m/z: 414 [M]+ (C29H50O).

Phytochemical investigation of D. pinnata (Poir.) Schumach. & Thonn. leaves indicated the
presence of various classes of secondary metabolites, including reducing sugars, cardiac
glycosides, coumarins, steroids, flavonoids, tannins, and alkaloids. The classes of compounds
were known to possess medicinal values. The compounds (1) and (2) belong to steroids as shown
in Figure 2. Therefore, D. pinnata (Poir.) Schumach. & Thonn. leaves containing these bioactive
principles is a good candidate for food and supplement even in daily drinks for medicinal
potential as an ant-stress.

Conclusion

It is rudimental information that phytochemicals of high relevance medicinally are yet exhorted
in natural products as the preliminary test in this work revealed. D. pinnata (Poir.) Schumach. &
Thonn. leave extracts revealed the presence of most tested phytochemicals. The isomeric
sitosterol isolated spectroscopically is known to have beneficial importance to the well-being of
humans in general, as good governance is achieved with good health. It is hoped that the food

9
and drinks industries will include these plant extracts in their preparation in form of ant-stress
supplements. this plant will have more future benefits to our immediate society (human) by
employing, advanced isolation techniques such as sensitive recycling HPLC and multiple radial
chromatography (RC) along with highly pure solvents to afford potential medicinal constituents,
being a catalyst to facilitate democratic government actors for excellent and efficient service
deliverance.

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Sitosterol from ethyl acetate extract of root bark of Terminalia. International Journal of
Scientific Research Publications, 5(3), 8–10.

Genser, B., Silbernagel, G., De Backer, G., Bruckert, E., Carmena, R., Chapman, M. J.,
Deanfield, J., Descamps, O. S., Rietzschel, E. R., Dias, K. C., & Mrz, W. (2012). Plant
sterols and cardiovascular disease: A systematic review and meta-analysis. European Heart
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Qian, K., Zheng, X. X., Wang, C., Huang, W. G., Liu, X. B., Xu, S. Di, Liu, D. K., Liu, M. Y., &
Lin, C. S. (2022). β-Sitosterol Inhibits Rheumatoid Synovial Angiogenesis Through
Suppressing VEGF Signaling Pathway. Frontiers in Pharmacology, 12(2), 1–13.
https://doi.org/10.3389/fphar.2021.816477

Rakel, D. (2018). Benign Prostatic Hyperplasia. In Integrative Medicine: Fourth Edition (Fourth
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Shi, C., Liu, J., Wu, F., Zhu, X. M., Yew, D. T., & Xu, J. (2011). β-Sitosterol inhibits high
cholesterol-induced platelet β-amyloid release. Journal of Bioenergetics and
Biomembranes, 43(6), 691–697. https://doi.org/10.1007/s10863-011-9383-2

Sundarraj, S., Thangam, R., Sreevani, V., Kaveri, K., Gunasekaran, P., Achiraman, S., &
Kannan, S. (2012). γ-Sitosterol from Acacia nilotica L. induces G2/M cell cycle arrest and
apoptosis through c-Myc suppression in MCF-7 and A549 cells. Journal of
Ethnopharmacology, 141(3), 803–809. https://doi.org/10.1016/j.jep.2012.03.014

Tovey, F. (2018). Duodenal Ulcer and Diet in Sub-saharan Africa. In Digestive Diseases in Sub-
Saharan Africa: Changes and Challenges. Elsevier Inc. https://doi.org/10.1016/B978-0-12-
815677-3.00008-6

Tripathi, N., Kumar, S., Singh, R., Singh, C. J., Singh, P., & Varshney, V. K. (2013). Isolation
and Identification of γ-Sitosterol by GC-MS from the Leaves of Girardinia heterophylla
(Decne). Open Bioactive Compounds Journal, 4(199), 25–27.
https://doi.org/10.2174/1874847301004010025

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Identification of Emerging Issues for Sustainable Development (pp. 77–94).

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 https://sustainabledevelopment.un.org/content/documents/10795Chapter5_GSDR2016.pdf

Bulama, J. S., M, D. S., & Mathias, S. N. (2015). Isolation and Characterization of Beta-
Sitosterol from ethyl acetate extract of root bark of Terminalia. International Journal of
Scientific Research Publications, 5(3), 8–10.

Genser, B., Silbernagel, G., De Backer, G., Bruckert, E., Carmena, R., Chapman, M. J.,
Deanfield, J., Descamps, O. S., Rietzschel, E. R., Dias, K. C., & Mrz, W. (2012). Plant
sterols and cardiovascular disease: A systematic review and meta-analysis. European Heart
Journal, 33(4), 444–451. https://doi.org/10.1093/eurheartj/ehr441

Kasote, D. M., Katyare, S. S., Hegde, M. V., & Bae, H. (2015). Significance of antioxidant
potential of plants and its relevance to therapeutic applications. International Journal of
Biological Sciences, 11(8), 982–991. https://doi.org/10.7150/ijbs.12096

Nguyen, J., Korta, D. Z., & Mesinkovska, N. A. (2019). Hair Supplements. In Alopecia. Elsevier
Inc. https://doi.org/10.1016/b978-0-323-54825-0.00028-4

Qian, K., Zheng, X. X., Wang, C., Huang, W. G., Liu, X. B., Xu, S. Di, Liu, D. K., Liu, M. Y., &
Lin, C. S. (2022). β-Sitosterol Inhibits Rheumatoid Synovial Angiogenesis Through
Suppressing VEGF Signaling Pathway. Frontiers in Pharmacology, 12(2), 1–13.
https://doi.org/10.3389/fphar.2021.816477

Rakel, D. (2018). Benign Prostatic Hyperplasia. In Integrative Medicine: Fourth Edition (Fourth
Edi). Elsevier Inc. https://doi.org/10.1016/B978-0-323-35868-2.00060-8

Shi, C., Liu, J., Wu, F., Zhu, X. M., Yew, D. T., & Xu, J. (2011). β-Sitosterol inhibits high
cholesterol-induced platelet β-amyloid release. Journal of Bioenergetics and
Biomembranes, 43(6), 691–697. https://doi.org/10.1007/s10863-011-9383-2

Sundarraj, S., Thangam, R., Sreevani, V., Kaveri, K., Gunasekaran, P., Achiraman, S., &
Kannan, S. (2012). γ-Sitosterol from Acacia nilotica L. induces G2/M cell cycle arrest and
apoptosis through c-Myc suppression in MCF-7 and A549 cells. Journal of
Ethnopharmacology, 141(3), 803–809. https://doi.org/10.1016/j.jep.2012.03.014

Tovey, F. (2018). Duodenal Ulcer and Diet in Sub-saharan Africa. In Digestive Diseases in Sub-
Saharan Africa: Changes and Challenges. Elsevier Inc. https://doi.org/10.1016/B978-0-12-

12
 815677-3.00008-6

Tripathi, N., Kumar, S., Singh, R., Singh, C. J., Singh, P., & Varshney, V. K. (2013). Isolation
and Identification of γ-Sitosterol by GC-MS from the Leaves of Girardinia heterophylla
(Decne). Open Bioactive Compounds Journal, 4(199), 25–27.
https://doi.org/10.2174/1874847301004010025

Wike, R., Simmons, K., Stokes, B., & Rhonda Stewart. (2017). Globally, Broad Support for
Representative and Direct Democracy. Pew Research Center, 1–43.
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