Phytochemical constituents of the roots of Heliotropium verdcourtii (Boraginaceae)

: The medicinal value of medicinal plants lies in some bioactive constituents that produce a definite physiological action on the human body. Heliotropium verdcourtii is a deciduous shrub or small tree traditionally used in the treatment of various diseases including fever, dry cough, measles, convulsions, epilepsy, diarrhea, and other ailments. The chemical constituents of the roots of the plant were not investigated to date. The aim of the study was to investigate the phytochemicals present in the roots of Heliotropium verdcourtii . The freshly collected root of the plant was chopped and air dried under shade. The dried and finely grounded plant root was extracted through maceration with n -hexane, chloroform/methanol (v/v 1:1), and methanol successively. The extracts were subjected to qualitative phytochemical tests for screening the classes of secondary metabolites present in the plant. Compound isolation of the chloroform/methanol (v/v 1:1) extract was performed through silica gel chromatographic separation. The structures of all isolated compounds were determined by spectroscopic methods as well as comparison with previous reports in the literature. The yields of n -hexane, chloroform/methanol (v/v 1:1), and methanol extracts were 2.2 g (0.4%), 25 g (5.0%), and 19.8 g (4.0%), respectively. The qualitative phytochemical test of the extracts revealed the presence of flavonoids, terpenoids, phenolics, saponins, glycosides and alkaloids. Silica gel chromatographic separation afforded a mixture of three isomeric triterpenoids identified as α-amyrin, β-amyrin, and bauerenol. To the best of our knowledge these bioactive compounds were isolated from the root of this plant, for the first time.


INTRODUCTION
The use of herbs and medicinal plants for primary human health care is a universal phenomenon.Today, as much as 80% of the people in the world depend on traditional medicine as primary health care (Kimutai, 2017).There is therefore need to investigate such plants to understand their chemical constituents.The genus Heliotropium being a small tree or shrub comprises about 40 species and belongs to the family Boraginaceae (Weigend et al., 2016).Many Heliotropium plants are mainly found being spread in tropical Asia, Africa, Australia, Europe, and Northern America (Gottschling & Hilger, 2004;Miller, 2003;Retief & Van Wyk, 2001).The bark, leaf juice, leaves, roots, seeds, stems, twigs and whole plant parts of Heliotropium are used as aphrodisiac, laxative, ethnoveterinary medicines, as traditional medicines, for ulcers and headaches, in treatment for schizophrenia, absorption of calcium, muscle protein, post-surgery recovery, sports injuries (Maroyi, 2021;Tidke et al., 2021).
H. verdcourtii is a plant highly distributed in Ethiopia, where it is locally named Game in Amharic, Hulaga, in Afan Oromo, and Gidincho in Sidama.It is a highly used as hedge plant in Ethiopia.Traditionally it is used for the treatment of various diseases like toothache, dysentery, tetanus, skin diseases and gastric ulcers (Chaluma et al., 2018).The decoction of the leaves of H. verdcourtii is used to improve the quality and quantity of milk products of livestock in Ethiopia (Bezabih et al., 2017).In Kenya an infusion and sap of the leaf is used to treat fever and as laxative agent, respectively.The root juice is used for healing wounds (Maundu and Tengnäs, 2005).
Despite the traditional use of the plant's roots against various life threatening diseases, there are few scientific reports dealing only with the phytochemical screening and biological activities of the root of H. verdcourtii.Recent reviews on the species, however, show that leaves of the plant are extensively studied, phytochemically (Maroyi, 2021).Hence this paper presents the results of the isolation and identification of chemical constituents from the roots extract of H. verdcourtii of Ethiopian origin.

Experimental
Grant thermostatic bath shaker (GLS-400) was used in the course of maceration of plant material.TLC spots were detected by a UV-2550 (SHIMADZU) UV-Vis spectrometer (Shimadzu, Kyoto, Japan).Column chromatography (CC) was performed with column size 3 cm × 30 cm packed with silica gel 60, size 0.063-0.200mm (70-230 mesh ASTM).Thin layer chromatography (TLC) was performed on aluminum sheets, silica gel 60 F254, and layer thickness 0.2 mm (Merck).NMR spectrum data was generated with 400 MHz for 1 H-NMR and 100 MHz for 13 C-NMR, TMS as internal standard and CDCl3 as solvent with the chemical shifts reported in parts per million (ppm).

Collection and Preparation of Plant Materials
H. verdcourtii specimens (leaves, flowers, seeds, stems) were collected on October, 2021 from Rufo Waeno Kebele, Aleta Chuko Woreda, Sidama Region, Ethiopia, for authentication of the plant.The plant was authenticated by botanist Retta Regassa Department of Plant Science, Hawassa College of Teachers Training, Hawassa, Ethiopia and a specimen was stored in the Herbarium of Hawassa College of Teachers Training with voucher no: HHF/0021-24.In addition to the aforementioned morphological parts, the roots were also collected for phytochemical analysis.The plant material was prepared in such a way that the root of H. verdcourtii was washed with tap water to remove soil particles and other foreign materials, and air dried in a shade for three weeks.The air-dried root was pulverized into powder using electric grinder.The pulverized plant material was kept in sealed plastic container and put on a dry cup board until used for extraction.

Extraction
The pulverized root (500 g) of H. verdcourtii was soaked in n-hexane (1.5 L) in a 5 liter Erlenmeyer flask, at room temperature.The flask was shaken for 72 h on an orbital shaker.The solution was filtered and the filtrate was concentrated under vacuum at 36 to 38 °C.The marc was further extracted with CHCl3:MeOH (v/v 1:1) and methanol successively, likewise.The extracts were put in refrigerator until used for further analysis.

Compound Isolation and Structure Elucidation
Solvent selection for the chromatographic separation was performed by various proportions of solvent mixtures of n-hexane, chloroform, dichloromethane, ethyl acetate, and methanol.The n-hexane-ethyl acetate solvent system showed better TLC profile and it was chosen for silica gel column chromatographic separation.Silica gel slurry of n-hexane was used for packing the column in order to achieve list polarity to the mobile phase.The CHCl3:MeOH (v/v 1:1) extract (15 g) was adsorbed on silica gel and was added to the column.Separation of the components through column chromatography was conducted with increasing polarity in n-hexane-ethyl acetate solvent systems of various proportions.22 fractions of each 30 ml were collected and the components in each fraction were analyzed by TLC.The fractions (on TLC) were concentrated using rotary evaporator.The concentrated and dried compounds were put in vial and stored in a refrigerator until sent for spectral analysis.Fraction number 11 (n-hexane: EtOAc; 4:1) resulted in a white solid compound (Rf = 0.57) with minor impurity.This was further purified by washing with n-hexane and resulted in 250 mg of white solid compound.The structure elucidations of the isolated chemical constituents were determined by generating 1 H-and 13 C-NMR data and by comparing the experimental spectroscopic data with previous reports in the literature.

Extraction Yield
Phytochemical investigation of specimens of plant origin is needed to increase the amount of chemical constituents and to maintain their activities (Aziz et al., 2003).Obtaining high extract yield is an important step in the course of secondary metabolite investigation and detection of biologically active compounds.Choice of appropriate extraction method is also essential for the tweaking of phytochemical constituents leaving out avoidable materials with the aid of the solvents.Further selection of suitable extraction process and optimization of various parameters are very important for up scaling from bench scale to large scale phytochemical analysis.The most commonly used extraction techniques include conventional techniques such as maceration, percolation, infusion, decoction, hot continuous extraction etc.In this study cold maceration technique is used.The extraction yield of the plant material is presented in Table 1.Extraction solvent choice needs to be based on the plant material matrix properties, chemical properties of the secondary metabolites, matrix-metabolite interaction, efficiency and desired properties (Ishida et al., 2001;Hayouni et al., 2007).The extractability of solvents depends on compound solubility in the solvent, the mass transfer and the strength of matrix interaction with heat and mass diffusion rate (Dhanani et al., 2017).The extraction solvent choice also depends on what natural compounds or classes of natural compounds one is looking for.In this study, however, the focus was performing total phytochemical analysis of the plant roots.The high yield of the CHCl3:MeOH (v/v 1:1) extract suggests that constituents in the plant specimen are moderately polar.

Phytochemical Screening
Plants of the genus Heliotropium are rich in bioactive constituents such as phenolic acids, lignans, flavonoids, nitrile glycosides, quinonoids, steroids, triterpenoids, and pyrrolizidine alkaloid (Jeruto et al., 2011;Li et al., 2008).The previous report on phytochemical screening of the leaf extracts of H. verdcourtii shows the presence of alkaloids, saponins, glycosides, terpenoids, anthraquinones, phenolics, and flavonoids (Ogundajo & Ashafa, 2017).In this study, the result of the qualitative phytochemical test of the extracts revealed the presence of flavonoids, terpenoids, phenolics, saponins, glycosides and alkaloids which is in agreement with previous works.
The classes of secondary metabolites found in the plant have the following biological activities.Flavonoids are known by antioxidative, free radical scavenging, coronary heart disease prevention, hepatoprotective, anti-inflammatory, anticancer and antiviral activities (Kumar & Pandey, 2013).Saponins are known for their biological activities such as antimicrobial, antifungal, anti-inflammatory, antiviral, antioxidant, anticancer, and immunomodulatory effects (Juang & Liang, 2020).Glycosides are known to possess remarkable therapeutic potential and pharmacological activities.Analgesic, anti-inflammatory, cardiotonic, antibacterial, antifungal, antiviral, and anticancer effects are some of the pharmacological activities (Soto-Blanco, 2022).Terpenoids have antimicrobial and antidiarrheal activities (Prashant et al., 2011).Phenolic constituents exhibit antibiotic, antimicrobial, and antidiarrheal activities (Jacob and Burri, 1996;Prashant et al., 2011).Alkaloids exhibit a wide range of activities.They are not only biosynthesized in nature against herbivores but also decrease bacterial or fungal influx (Adamski et al., 2020).They are therefore constituents that have high prospective in medicine, plant defense, veterinary, or toxicology.The presence of such classes of secondary metabolites supports the ethnomedicinal use of the species.

Isolated Compounds and Their Structure Elucidation
Fractionation of the chloroform:methanol (v/v 1:1) extract resulted in a white solid compound with Rf = 0.57 (n-hexane-EtOAc 4:1).The 1 H-, 13 C-NMR, and DEPT-135 spectral data, however, revealed the white solid compound being a mixture of three compounds (isomers) where purification through silica gel column chromatography and recrystallization was not successful.The experimental spectroscopic data was compared with spectroscopic data reported in literature (Carothers et al., 2018;Chaluma et al., 2018;Liliana et al., 2012;Mesfin, 2018;Raga et al., 2013;Sathish et al., 2017) for structure elucidation.

Compound 2
The 1 H-and 13 C-NMR spectral data of this compound is similar to compound 1 except some differences in the 13 C-NMR spectra.The 13 C-NMR spectra peaks at δ 145.3 and 121.7 belong to the olefinic carbons (C-13 and C-12) and δ 40.0 and 37.7 are C-20 and C-19 respectively.The difference in the chemical shift of the C-20 and C-19 is due to the shift of the methyl groups towards C-20 which resulted in an increase in C-20 and a decrease in C-19 chemical shift values.The chemical shift at C-3 also showed a slight shift and appeared at δ 79.0 which also distinguishes this compound from compound 1.
Ursane type triterpenes are widely distributed in the plant kingdom, as aglycones or in combined forms, and have several biological activities.α-amyrin is usually found in oleo-resin of the various species of Bursera or Protium of the Burseraceae family.It exhibits several biological activities in vitro and in vivo conditions against several health-related conditions, such as microbial, inflammation, cancer cells, and viral and fungal infections (Liliana et al., 2012).Bauerenol, on the other hand, showed cytotoxic and apoptotic potential against human HepG2 cancer cells and it is also anti-Trypanosoma brucei agent (Carothers et al., 2018).In addition to this bauerenol prevents migration, proliferation and invasion of retinoblastoma cells through induction of autophagy, apoptosis and cell cycle arrest (Chen et al., 2022).β-Amyrin possesses anti-inflammatory, anti-fibrotic, and anti-apoptotic effects on dimethyl nitrosamineinduced hepatic fibrosis in male rats (Thirupathi et al., 2017).The presence of α-amyrin might have caused the root of the species to have the traditional medicinal effects as wound healing (Maundu & Tengnäs, 2005).

CONCLUSION
In this study, phytochemical screening and compound isolation were carried out on the root of H. verdcourtii.The classes of secondary metabolite screening test of the n-hexane extract revealed the presence of terpenoids, flavonoids, and glycosides, alkaloids, saponins, and phenolics.The presence of these bioactive constituents is significant as they may account for the wide scope ethnomedicinal use of the species.Silica gel column chromatographic separation of the chloroform/methanol (v/v 1:1) extract has led to the isolation of the mixture of three biologically active ursane type pentacyclic triterpenes identified as α-amyrin, β-amyrin, and bauerenol.The presence of α-amyrin might be the cause of the root to have a wound healing property.This is the first report of the isolation of the aforementioned chemical constituents from the root of H. verdcourtii, of Ethiopian origin.

Table 1 .
Percent yield of the crude extracts.