Analysis of the Phytochemical Constituents of Rauvolfia caffra to Ascertain its Sedatives Potentials

By Onu B. (2023)

Greener Journal of Life Sciences

ISSN: 2384-633X

Vol. 5(1), pp. 1-11, 2023

https://gjournals.org/GJLS

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Analysis of the Phytochemical Constituents of Rauvolfia caffra to ascertain its Sedatives Potentials

Benefit Onu (PhD)

Federal University Otuoke, Faculty of Science, Biology Department

ARTICLE INFO

ABSTRACT

Article No.: 081023081

Type: Research

Full Text: PDF, HTML, PHP, EPUB, MP3

This research focuses on the analysis of phytochemical constituents and the evaluation of sedative potentials of Rauvolfia caffra. The phytochemical constituents as well as its sedative potentials and most medicinal values of Rauvolfia caffra were evaluated in this work using standard laboratory analytical techniques. This study revealed that the percentage composition of alkaloids (13.37%), tannins (0.28%), saponins (6.30%), phenols (3.30%) in the root were significantly higher (p<0.05) when compared to their composition (concentration) in the leaves and stem bark. The percentage of terpenoids (1.73%) and steroids (1.24%) in the stem bark were significantly higher (p<0.05) when compared to their composition (concentration) in the leaves and root. The percentage of flavonoids (8.32%) in the leaves were significantly higher (p<0.05) when compared to its composition (concentration) in the stem and root. The result also shows that sedative potentials of Rauvolfia caffra analyzed from the root have higher percentage of alcohol (31%) when compared to the stem bark (0.29%), while the stem bark of Rauvolfia caffra has a higher content of barbiturate (5.30mg) when compare to the root (0.52mg). This research suggests that with the presence of barbiturate and alcohol in the sample evaluated in the laboratory, it implies that Rauvolfia caffra has sedative potentials that can be extracted and utilized as sedative drug in our hospitals. Therefore, massive propagation and conservation of Rauvolfia caffra to enhance the production of sedative herbal modern medicine should be encouraged.

Accepted: 13/08/2023

Published: 23/08/2023

*Corresponding Author

Dr. Benefit Onu

E-mail: Benefitonu28@ gmail.com

Keywords: Phytochemical constituents, Sedative potentials, Rauvolfia caffra.

INTRODUCTION

In using herbal medicine against disease, it is possible to limit side effects that manufactured synthetic drugs from the pharmaceutical industry can induce (Briskin, 2000; Lee and Bae, 2017). Available synthetic medicines and their generic forms are expensive, especially as poverty is an increasing concern globally and affects more than five billion people in developing countries (Afzal et al., 2011; Hoang et al., 2017). Naturally available flora, provides inexpensive treatments that benefits a large portion of the global community (Afzal et al., 2011; Amuka et al., 2017).

Since prehistory, plants have been used extensively as medicine for the treatment of various ailments, even today this trend continues. According to WHO, approximately 75-80% of the world’s population use plant-based medicines. All plants may not be as useful as claimed, or may have more therapeutic properties than are known traditionally. Therefore, proper scientific knowledge is required to investigate and explore the exact standardization of such medicinally important plants.

Traditional medicine systems form a large part of the health expenditure in Some African Countries. In South African and other Sub-saharan African countries, about eighty percent of the population groups consult traditional healers regularly while other population have specific cultural traditions (Tetyana et al., 2002; Afolayan and Lewu, 2009; van Vuuren, 2008; Xego et al., 2016; Petersen et al., 2017). Of the thirty thousand plants that make up the distinctive floral diversity, only about three thousand species are employed in a therapeutic manner (van Vuuren, 2008; Xego et al., 2016). The scientific information on these medicinal plants is scarce amidst the considerably recorded written reports on the relationship between the people and the plants they choose to use (Hutchings et al., 1996; van Vuuren, 2008). The indigenous medicinal plants of African countries like Nigeria offer a wide array of natural plant compounds for the purposes of treating common illnesses that are prevalent in third world countries (Moteetee and Kose, 2017). The need for confirmation and approval by the scientific method comes from the heavy dependence of the destitute to utilize inexpensive forms of medication (Hutchings et al., 1996; van Vuuren, 2008). In the standard functioning of plants, secondary metabolites or phytochemicals are formed which is not vital in the survival of the plant but may have other functions (Okigbo et al., 2009). Importantly for humans, plants also have compounds that serve as therapy or healing for common and fatal human illnesses such as cancer, tuberculosis and malaria (Ramamurthy and Sathiyadevi, 2017). Existing research has shown that about half of all medicine available commercially is from plants or derived from plant sources.

Many illnesses have come to light in recent years. These diseases are responsible for a large percentage of deaths in African countries. (Motsei et al., 2003). Natural sources have been effective in providing cures for most of these illnesses (Boadu and Asase, 2017; Doffana, 2017; Kinda et al., 2017). According to Olukayode et al., (2011) and Ginovyan et al., (2017), it is imperative to institute the regulation and reliability of customary medical care, which highlights the importance of testing the antibacterial and antimicrobial activities of the plant species used.

In the Kingdom Plantae, microscopically distinguishable plant structures carry out the activities of either secreting or excreting biochemical substances (Cutter, 1978; Samuel et al., 2018). Secretory components may occur in various locations in plant tissues and cells. By characterizing and identifying secretory structures, distinguishing features may arise and in only a particular family of plants (Cutter, 1978). The cytoplasm may be the site of the production of the substance and its transportation towards the outside of the cell is defined as ‘secretion’ (Cutter, 1978). Excretion on the other hand refers to the substances that the plant has no benefit from (Cutter, 1978). The substances secreted by plants have potential to be used for medicinal and economic gain (Cutter, 1978). The medicinal components of plant tissue can be found in oil glands and glandular structures of leaf hairs, also called trichomes. The latter are thread-like outgrowths that originate from the epidermal layer of plant tissues (Levin et al., 1973; Barthlott et al., 2017). They occur in many different conformations across the surfaces of leaves, stems and roots. They are also responsible for carrying out a variety of fundamental functions required by the plant to adapt and thrive in its environment ( Elzbieta and Chernetskyy. 2005; Barthlott et al., 2017).

Generally, trichomes function to enhance wind protection to prevent the epidermal layer from dissipating water from the tissues, decreasing the absorbance of sunlight and increasing the absorption of moisture (Levin et al., 1973; Dalin et al., 2008). The secretory structures present in plants serve as a vital component of indicating the presence of phytochemicals, secondary metabolites and other secretions that form part of the plants biochemical defense system (Umah et al., 2017). Effectively, the biological activity of the phytochemicals determines the degree of pharmacological assistance (Umah et al., 2017).

It is well known that plants from the Apocynaceae family contain latex and have been used medicinally (Lopes et al., 2014). Similar to other Apocynaceae species, Rauvolfia caffra has characteristic white latex which is used to treat stomach problems (Mnxati, 2011). This plant has been used traditionally for its medicinal benefit; however, there is scarce information on its sedative potentials. Rauvolfia caffra has been used traditionally in the treatment of sexually transmitted infections and general health problems such as skin infections, fever and pneumonia (Njau et al., 2014).

Sample Description: Rauvolfia caffra

Rauvolfia caffra is a plant species belonging to family Apocynaceae. The Apocynaceae is one of the largest families with 300 genera and around 5000 species (Endress, 2004; Endress et al., 2014). It is commonly known as “quinine tree and is widely used in Africa by natives as a medicine (Freiburghaus et al., 1996). The family comprises large tree, shrubs, wild herbs and even vines (Freiburghaus et al., 1996). In Africa R. caffra is widely distributed in riverine Branchiostegal woodlands, lowlands, in dry montane rainforests and in swamps (FAO, 1986). R. caffra (Apocynaceae) are rich in indole chemical most of which have been isolated and identified in several literature.

Qualitative Phytochemical Screening of Rauvolfia caffra

Test for Alkaloids

0.2g of dried and powdered leaves were boiled in a boiling tube with 5ml of 2% of HCL on a steam bat for 5min. The mixture was filtered after cooking. The filtrate was divided into 3 test tubes A, B, C. Test A 1ml portion of filtrate was treated with 2 drops of Mayer’s reagent, a creamy white precipitate was observed. This was confirmed with 1ml of filtrate treated with 2 drops of Dragendroff reagent which gives a red precipitate to indicate the presence of alkaloids.

Test for Tannins

To 2g of the powdered samples 5ml of 45% ethanol was added and boiled in a water – bath for 5min. The mixture was cooled and filtered. 1ml of the filtrate, 3 drops of lead acetate of solution were added. The formation of gelatinous precipitate indicates the presence of tannins. Also, as a confirmation test, 1ml of the filtrate was treated with 0.5ml of Bromine water and the formation of a pale brown precipitate indicates the presence of Tannins.

Test of Flavonoids

0.5g of powdered sample was introduced into a boiling tube, 10ml of ethyl acetate was added and the mixture was brought to boiling in a water bath for 1min. The mixture was cooled and filtered, 4ml of the filtrate was treated with 1ml of aluminum chloride (ALCL₃) solution (1%) and left to stand for 10min. The formation of a yellow coloration in the presence of 1m of chloride ammonium solution (hydroxide) indicates the presence of Flavonoids.

Test for Saponins

0.2g of powdered sample were boiled with 5ml of distilled water in a boiling tube in a water-bath. The mixture was filtered while still hot. 1ml of the filtrate was treated with 3drops of olive oil and the mixture vigorously shaken. The formation of an emulsion was observed. Another 1ml was shaken with 1ml of distilled water and the formation of a stable frothing on standing indicated the presence of saponins.

Test for Cardiac Glycosides

2g of powdered sample was boiled with 30ml of distilled water for 5min. The mixture was cooled and filtered. 5ml of the filtrate, 0.2ml of Fehling’s solution A and B were added and boiled for another 3min. A brick red coloration indicates the presence of glucosides.

Test for Terpenoids (Salkowski Test)

0,5g of sample was treated with 2ml of chloroform, and 3ml of conc H₂SO₄ was carefully added to form a layer. A reddish-brown coloration of the interface indicates the presence of terpenoids.

Test for Phenols

1ml of the aqueous filtrate was treated with 3 drops of ferric chloride solution. The formation of a blue-black color indicates the presence of phenol. Also, 1ml of the filtrate was treated with 3drops of lead acetate solution. The formation of a yellow-colored solution indicates the presence of phenols.

Quantitative Phytochemical analysis of Rauvolfia caffra

Determination of Alkaloids

5g of powdered samples were placed in a 250ml beaker, and 200ml of 10% acetic acid was added and covered with aluminum foil and allowed to stand for 4hours. This was filtered and the filtrate concentrated to about ¼ of its original volume on a water-bath. Conc Ammonia solution was added drop-wise to the filtrate until precipitation was completed. The solution was allowed to settle. The ppt was then collected over a Whatman No 1 filter paper and further washed with dilute ammonia solution. The residue was dried in the oven at 65⁰C until completely dried, it was then weighted as the alkaloids was obtained.

% Alkaloids = b/a x 100/1

Where: a= weight of sample

b= weight of dried ppt

(Harborne, 1998)

Determination of Tannins

0.5g of sample was weighted into a plastic bottle (100ml) and 50ml of distilled water was added and shaken for 1hr on a mechanical shaker. This was filtered into a 50ml volumetric flask and the solution made up to the mark. 5ml of the filtrate was transferred in the test-tube and mixed with 2ml of 0.1m Fecl₃ in 0.1m Hcl and 0.008m potassium ferro cyanide. The spectrophotometer was set at 220nm. The absorbance was measured within 10min. Tannic acid was used to plot the stand curve.

Determination of Saponins

2.0g of powdered samples were put into a conical flask and 100ml of 20% Ethanol was added. The samples were heated over a water bath for 4hrs with constant stirring at 55⁰C. The mixture was then filtered and the residue re-extracted with another 100ml of 20% ethanol. The combined extracts were reduced to 40ml over a water bath at 90⁰C. The concentrate was transferred into a 250ml separating funnel and 20ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification was repeated. 60ml of n-butanol was added. The combined n-butanol extract was washed twice with 10ml of 5% NaCl solution. The resulting solution was evaporated on a water bath to a constant weight. The saponin content was then calculated.

Determination of Flavonoid

This method is based on the formation of the Flavonoid-Aluminum complex which has an absorptivity maximum at 415.100ul of the sample extract in methanol (10mg/ml) mixed with 100ul of 20% Alcl₃ in methanol and a drop of acetic acid and then diluted to 5ml. The absorbance at 415 was taken after 40min to develop the color. A blank was prepared from 100ul of sample extract and a drop of acetic acid and diluted to 5ml with methanol. The absorbance of routine (std for flavonoid) solution 0.5mg/ml in methanol was measured under the same condition.

Estimation of Barbiturate

To estimate the amount of barbiturate present in the samples, A Perkin–Elmer Lambda 6 and Spectronic 21D were used for spectrophotometric analysis with 1 cm quartz cells. 2.2. Assay method utilizing spectrophotometer and micro reaction tubes at an absorbance of 530 nm wavelength with UV–V spectrometer (Bartzatt, 2002). Bartzatt, (2002) steps were then used for comparison and interpolating concentrations of test samples.

Estimation of alcohol

The estimation of alcohol was based on the complete oxidation of ethanol by dichromate in the presence of sulfuric acid with the formation of acetic acid. This reaction is highly preferred because potassium dichromate is easily available in high purity and the solution is indefinitely stable in air. The reaction that occurs between alcohol and potassium dichromate is: 2Cr₂O₇– + 3C₂H₅OH + 16H+ 4Cr+++ + 3CH₃COOH + 11H₂O.

Figure 8 shows the sedative content of the extracts of R. caffra. This result revealed that the roots of Rauvolfia caffra have a higher percentage of alcohol (31%) when compared to that of the stem bark (0.29%). The stem bark of Rauvolfia caffra had a higher content of barbiturate (5.30mg) when compared to that of the roots (0.52mg).

DISCUSSION OF FINDINGS

Natural remedies and medicinal plants are a great source of bioactive chemicals that can be exploited to find novel therapeutic agents and treat a variety of disorders (Nesa et al., 2018). Phytochemicals occurring naturally in plants can have either positive or negative impacts on human health. The richest bio-reservoirs of different phytochemicals are found in medicinal plants that are used to treat various illnesses and conditions. The phytochemical components of plants determine their therapeutic qualities. Alkaloids, flavonoids, phenolics, tannins, saponins, steroids, glycosides, terpenes, and other essential phytochemicals are found throughout various plant parts (Nortjie et al., 2022; Shaikh and Patil, 2020).

Crude extracts of R. caffra’s leaves, stem bark, and roots were screened for phytochemicals, and the following groups of substances were discovered: alkaloids, tannins, terpenoids, saponins, glycosides, flavonoids, phenols, steroids (Table 1 and 2). For each class of substance, these findings held true despite minor variations in abundance. The R. caffra extract phytochemicals content results further demonstrate their therapeutic benefit, making them potential agents in preserving the anti-oxidant, anti-inflammatory, antiviral, anti-hypertensive, anti-diabetic, anti-malarial, anti-microbial, analgesic, and anti-carcinogenic properties in living systems (Egbuna and Ifemeje, 2015; Forni et al., 2019). The presence of antioxidant-active compounds in the R. caffra samples was confirmed by the detection of alkaloids, terpenoids, saponin, cardiac glycosides, and steroids. These results confirm the importance of using indigenous knowledge to identify plants for medicinal purposes. The presence of phytochemicals with documented health advantages supports the use of quinine trees in traditional medicine. R. caffra contains cardiac glycosides, which may be the reason why the plant has historically been used to treat heart conditions (Milugo et al., 2013).

The results from the qualitative screening of the extracts of R. caffra correspond with the previous reports from this plant and other species of Rauvolfia (Deshmukh et al., 2012; Ebeh Messanga et al., 2018; Jamkhande et al., 2013; O’Connor and Maresh, 2006).

According to (Tlhapi et al., 2018), high concentrations of reserpine, ajmaline, and ajmalicine, three well-known alkaloid chemicals, can be found in the roots and stem bark extract of R. caffra, and this corresponds with the findings from this study. Species of Rauvolfia are frequently used to treat a variety of illnesses. Due to the presence of alkaloids, extracts of Raulvolfia are found to be effective in treating neuropsychiatric patients' schizophrenia. They also have a sympatholytic effect and are frequently used to treat hypertension, corneal opacities, epilepsy, skin conditions, chest pains, internal disorders, hair loss restoration, and convulsions (Kadiri and Ayodele, 2021).

The high amount of alcohol and barbiturate reveals the sedative abilities of R. caffra. Alcohol has been proven to exhibit sedative effects (Chung and Martin, 2009; Hendler et al., 2013). Alcohol has a stimulating and a sedative effect on people (Hendler et al., 2013). Alcohol specifically inhibits executive cognitive functioning, a group of higher-order cognitive skills like organizing, planning, abstract reasoning, cognitive flexibility, and monitoring one's own and other's behaviour (Hendler et al., 2013). Barbiturates are a class of sedative-hypnotic drugs that are used to treat seizures, newborn withdrawal syndrome, insomnia, preoperative anxiety, and inducing comas in the event of high intracranial pressure. For causing anesthesia, they are also helpful (Skibiski and Abdijadid, 2022). In both outpatient and inpatient settings, barbiturates have historically been a class of medications that are frequently administered. Barbiturates are used therapeutically as intravenous anesthetics, antiepileptic medications, and sedatives or hypnotics. Due to their negative effects, including drowsiness, addiction, and worsening of seizures upon withdrawal, barbiturates are not commonly used nowadays. However, phenobarbital and other barbiturates are still been used as anticonvulsant medications (Maideen, 2019). When barbiturates and alcohol are taken by a patient, the risk of sedation is increased. The complications brought on by mixing alcohol and barbiturates must be explained to the patients (Maideen, 2019). As a result of this, the use of R. caffra increases the risk of sedation in the users, because of its high barbiturates and alcohol content.

Sedatives are used to treat anxiety, nervous tension, pain, and to aid in sleep when there is insomnia (Nkundineza et al., 2020). R. caffra may exhibit sedative effects as a result of the presence of barbiturate, which can also be linked to the presence of alkaloids and flavonoids in its extracts (Nkundineza et al., 2020), and these can be used as basic medicinal agents for their analgesic, antispasmodic effects. The roots of R. serpentina (a species of Rauvolfia) are used as a treatment for a variety of ailments in Ayurvedic medicine, including hypertension, insomnia, mental agitation, gastrointestinal issues, excitation, epilepsy, trauma, schizophrenia, sedative insomnia, and insanity.

CONCLUSION

As a result of the presences of alcohol and barbiturate as well as phytochemicals such as alkaloids, flavonoids, tannins, saponins, glycosides in the root, stem bark and leaves of Rauvolfia caffra, this research considered Rauvolfia caffra as a viable herbal choice in pharmaceutical industry to be used as a raw material for the production of sedative medicines. Extracts of Raulvolfia caffra obtained from its roots and stem barks contained more concentrations of phytochemicals and sedative parameters and as a result, may be suitable for medicinal purposes and to treat a variety of ailments such as hypertension, insomnia, mental agitation, gastrointestinal issues, excitation, epilepsy, trauma, schizophrenia, sedative insomnia, insanity, and various nervous disorders. The ethnomedicinal uses make it one of the most important medicinal plants used in the suppression of skin diseases and infections. The presence of phytochemicals confirms R. caffra's historical therapeutic use and demonstrates that traditional medicine is a reliable source of information for the creation of novel medications. The use of sedatives should also be monitored as extensive usage can lead to addiction and drowsiness. Massive propagation of rauvolfia caffra should be embarked upon to enhance sedative herbal and modern medicine. In order to obtain a complete picture of its in vitro sedative effects in living organisms, additional study should be conducted to extract, identify, define, and elucidate the structures of more of the bioactive chemicals present. Extensive studies should be carried out on the plant so as to determine more medicinal properties of Raulvolfia caffra. There is also the need to discover more domestic ways to increase its production and improve its preservation.

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	%Alkaloids	%Tannins	%Saponins	%Flavonoids	%Terpenoids	%Phenols	%Steroid
Leave	2.86±0.02*	-	3.97±0.01*	8.32±0.01*	0.30±0.02*	0.42±0.02*	0.02±0.02*
Stem bark	3.76±0.02*	0.25±0.02*	1.86±0.02*	3.54±0.02*	1.73±0.02*	0.48±0.02*	1.24±0.01*
Roots	13.27±0.02*	0.28±0.002*	6.30±0.02*	6.76±0.01*	1.58±0.02*	3.30±0.02*	0.65±0.02*

INTRODUCTION

Sample Description: Rauvolfia caffra

MATERIALS AND METHODS

MATERIALS

Description of Study Area

Collection of Plant Samples