Phytochemical Screening of Cit

Phytochemical Screening of Citrus nobilis (Dalanghita) peel
Myra M. Ronquillo, A Requirement in partial fulfilment of the subject Chemistry of Natural Products. Institute of Graduate Studies, Central Luzon State University, Science City of Muñoz, Nueva Ecija

Abstract

This experimental study was conducted to perform phytochemical screeningcitrus nobilis (dalanghita) peel.
This study made use of the experimental research design in an actual laboratory set-up. There were two phases in the experimental study. Phase 1 included the preparation of the plant sample and extraction using denatured alcohol, Phase 2 is the phytochemical screening to determine the presence of reducing sugars, anthraquinones, terpenoids, flavonoids, saponins, tannins, alkaloids, and cardiac glycosides in the peel extract.
Findings showed that dalanghita (Citrus nobilis) peel extract contains reducing sugars, terpenoids, alkaloids, cardiac glycosides, flavonoids and tannins. These substances can become excellent sources of therapeutic substances and as additive to cosmetic products. Based on the findings, the following recommendations were drawn: A follow up study should be conducted to quantify, isolate and identify the type of reducing sugars, terpenoids, alkaloids, cardiac glycosides, flavonoids and tannins present in the peel of dalanghita. tests on the presence of other compounds are highly recommended, to further explore the potential of dalanghita peel as a source of alternative drug and other industrial applications, pharmacological testing should be done, and other parts of dalanghita should be used for phytochemical screening and bioassays.

Key words: Citrus nobilis, Phytochemical screening, secondary metabolites

INTRODUCTION

Long before the development of methods in verifying active components in plants, the use of herbal medicine has been a practice among Filipinos. Without knowing exactly the science behind the claims, people of the rural areas rely mainly on herbolaryos or quack doctors for remedy. The trend has subsided with the rise of advanced technology, introduction of science-based medicine, the trust of the patients for their health concerns has shifted to the expertise of medical doctors.

Today, there is a paradigm shift on the use of alternative medicines, the reason being mainly on the side effects and high cost of medicines, let alone the development of resistance among pathogenic organisms. Secondary metabolites, though present in plants and animals in small amounts and for defense mechanisms, have received a great deal of attention in the medical world. Many drugs have been discovered through research into the physiological effects of chemicals found in plants. Some of these drugs are still derived directly from plants (e.g. digitalin from foxglove, Digitalis) while others are now synthesized (e.g. aspirin inspired by the medicinal properties of the bark of the willow, Salix alba). Chemicals in some plants are extracted and transformed, providing the building blocks of drugs (e.g. progesterone, synthesized from chemicals found in some species of Dioscorea, the wild yam (BGCI Fact Sheet, 2000).

As a preliminary step, phytochemical screening is geared towards the discovery of the array of potentials a species holds.

Objectives

The study aimed to perform phytochemical screening of dalanghita, citrus nobilis, peel.

Scope and Delimitation

The study was delimited to the phytochemical screening dalanghita, Citrus nobilis, peel. Only the peel were used in the experimental investigation. The fruits were gathered from Brgy. Bangkal, Abucay, Bataan.
The determination of the chemical constituents was limited to the qualitative rather than the quantitative analysis. Only the presence or absence of the secondary metabolites alkaloids, quaternary bases or amine oxides, saponins, free fatty acids, cardiac glycosides, leucoanthocyanins, flavonoids, tannins, fats and anthraquinones were given focused to.

Review of Related Literature
This section includes a summary of readings and studies of both local and foreign researchers which are relevant to the study.

The Quest for Alternative Medicine
Guevara and Recio as cited by Reotutar and Supnet, (2007) mentioned that there has been in more recent times an awakening towards the use of drugs and their preparation in a kind of “back-to-nature” movement, instead of the classical synthetic compounds manufactured in advanced countries. While the use of synthetic drugs is of undoubted value, especially in advanced state of illness, it is believed that the use of herbal medicines of properly tested efficacy would be of great advantage in a developing country like the Philippines which is blessed with bountiful plant resources. The idea is to keep people healthy by treating illnesses at an early stage instead of resorting to treatment when the disease is already at an advanced stage.
The importance of herbal plants offer alternative remedies with tremendous opportunities. They not only provide access and affordable medicine to poor people. They can also generate income, employment and foreign exchange for developing countries. People who live in rural areas of the Asia Pacific are familiar with the medicinal properties of plants, growing close to their homes, in the open fields, water margins, waste lands, both inside and outside the nearby forest areas and under different growth conditions. Most of the plant materials collected used are fresh, either to obtain the extract from the whole plant or parts thereof, whether they be leaves, roots, flowers or fruits. In case of woody forms, mostly the bark, roots and other parts are used. Carminatives like ginger, cloves and coriander are also usually added as fresh or dried materials. Though dried plant parts are frequently used, often the easy availability of fresh material is a critical point and the herbal doctor in the village is well familiar with various plants he needs, their growth patterns, seasonality, habitat and other details. Such details were usually passed on in the past from parent to offspring in the family and uses of plants and the various combinations or mixes made were kept as a family secret. Along with the development of knowledge at family level, tremendous progress has been made at using the plant products at professional level in different societies, which have grown into branches of science in their own right. Most of the methods and uses were taught orally and through demonstration and very few records or writings were maintained. Such professional practices are continuing even today, As villagers migrated to the city, losing touch with past practices or when there was no heir apparent to the village doctor, the precious knowledge is usually lost. (Amanonce et.al., 2007)
Traditional medicine has to be considered as the cost of health care has become unaffordable to a great majority of the population. A big segment of the population believe in traditional cures, yet we have not documented its effectiveness. In the Region, we should first come up with the profile of the different modalities of treatment and determine their acceptability. Further, there is a need to define traditional medicine. (Cayabyab, 2004)
Funded programs of the Department of Science and technology is aimed at intensified research on indigenous plant materials not only for drug manufacturers but also primarily for providing the rural areas with adequate supply of medicines or drug preparations by the expanded utilization of the plants in their raw and semi-processed forms.
Primary screening provides a general profile of the toxicity, pharmacological activities and pharmacokinetics of a new drug. The results obtained with the animal models are used to evaluate the safety of the material, its toxic effects and its intended therapeutic properties. Thus, it is essential that the pharmacological and toxicological properties of the drug material be established before any clinical trials on man are conducted.
Tabudlo (1996) conducted phytochemical analysis on the leaves and stems of hagonoi plant (Chromolaena odorata). It was found that the aqueous and alcoholic leaf extracts of hagonoi plant contain glycosides, saponins and tannins. Sterols were extracted only from the aqueous medium while terpenes were only extracted from the alcoholic medium. Further, the study also found out that there are coagulating effects of the aqueous and alcoholic leaf and stem extracts of the hagonoi plant using male Swiss mice as test animals.
This study is different from Tabudlo’s scientific investigation in that, though both studies utilized the same plant, this study concentrated more on the leaves of wellawel. Morever, antimicrobial assay was also conducted to determine of the leaf extract has an inhibitory effect towards the growth of certain types of microorganisms. Crude extract and aqueous extract of the leaves were also used to test the coagulatory effects of the leaves on Swiss mice.
Viado (2006) performed phytochemical, mirobiological screening and pharmacological testing on water hyacinth ( Eichornia crassipes Linn) and kataka-taka (Kalanchoe pinnata Linn). Results of the phytochemical screening indicated that the water hyacinth contained flavonoids, tannins and resins in the leaves and the kataka-taka leaves contained alkaloids, saponins, flavonoids and resins. The Kirby Bauer Disk Diffusion method showed that the kataka-taka crude leaf extract possesses an active inhibitory activity against Staphylococcus aureus and partial inhibitory activity against Escherichia coli and Candida albicans. The water hyacinth did not manifest inhibitory activity against bacteria and fungi test organisms.
The water hyacinth and kataka-taka also showed a positive coagulating effect as shown by the 48 seconds and 82.75 seconds coagulation time compared to the 82.75 seconds coagulating time of the positive control group and 93.25 seconds coagulation time of the negative control group.
This study is basically similar with Viado in that both studies perform phytochemical, microbiological screening and tested the coagulating properties of the said plant. They only differ in the plant specie used.
Rabena (1999) conducted the isolation, characterization and identification of the active components of kakawate leaves (Gliricidia sepium Jacq. Kinth.Ex.Walph) against termites. Chopped kakawate (Gliricidia sepium Jacq.Kinth. Ex. Walph) were soaked in petroleum ether. Crude extract recovery corresponded to 5.35% of the total fresh weight of the leaves. Feeding the termites with petroleum ether crude extracts at 0.02 g/ml gave 100% mortality rate within four hours and 100% within 8 hours. One hundred termite mortality is observed four hours after feeding 0.02 g / ml (20,000 ppm) purified coumarin. Thin layer chromatography of the petroleum ether crude extract gave two major fractions. Chromatography on a column packed with silica gel 60 and elution 4:6 petroleum ether; chloroform yielded a pure compound, C9H6O2, which accounted for 2.83% of the leaf fresh weight. It was characterized by UV, IR, MS, 1H NMR and 13C NMR.

Dalanghita: Exploring the Possibilities
Dalanghita, popularly known as sintores in the province of Bataan, is a small tree. The leaves are smooth, oblong to broadly lanceolate, 4 to 10 cm long, with short petioles of about 1 cm long. A flowering plant, its flowers are white, solitary and short-pedicelled. The fruits are hesperidums, with a loose skin and leathery pericarp, with a sweet pulp that is only fairly juicy of many varieties, the large ones attain a size of about 10 cm.
It is widely cultivated in the Philippines for commercially fruiting, the fruit bearing period is at its peak during the months of October to February. The fruit is a good source of vitamin A, B and C, and Citric acid which is 0.35% of the juice, and the rind is used for flavouring. Studies on the composition of the rind revealed the presence of 14-19.33% volatile oil, 92% limonene, methylanthranillic acid, and methyl ester Both seeds and cuttings are used for propagation.
Rind is prepared by gathering the rind ripe fruit which is sun-dried, and can be used as either whole or cut into thin slices. The seeds are placed in a container, where an amount of water is added to one fiftieth of the total weight of the seeds. This is stored for a short time and put into a dry kettle. It is then heated with a weak fire until the materials turn light yellow and smell fragrant. The material is sundried and crushed before it could be used.
This seed preparation has pain relieving effect. In Malaya, a decoction of the roots is used in dysentery. Powdered leaves with leaves of Areca catechu may be drunk for stomach ache. An infusion of the fresh juice is used as a cleanser or stimulant of wound surfaces. A lotion of the boiled leaves is used hot on painful places and swelling in Malaya. Seed preparation tastes bitter with pain relieving effect. Rind preparation tastes bitter, with mild nature. The fibers of the rind tastes bitter-sweet and neutral natured. The rind is also used for nausea and fainting. It is squeezed near the nostril for instant inhalation. Decoction of roots is used for cough and fever.
For anorexia and vomiting, fresh rhizome of ginger is added to a concentrated decoction of the rind and this decoction is drunken. Decoction of dried flowers is used for diarrhea.
Decoction of rind or peel used to regulate monthly period.
Leg bath of boiled leaves used for rheumatism, and painful and swollen legs.
The oil from the rind is used for stomach problems; and as liniment for gout, rheumatism and other painful swellings.
Phytochemical screening of the volatile compounds in three selected Asian citrus fruits (C. nobilis, C. sinensis, C reticulata) revealed a total of 51 compounds in C. nobilis: terpenes, carbonyls, alcohols, esters and hydrocarbons, with limonene as the main compound.
Naringin, a prominent bioflavonoids in grapefruit and other citrus fruits, is present in C. nobilis. Study on the free radical scavenging of naringin revealed a dose-dependent scavenging and demonstrates it can protect mouse bone marrow cells against radiation-induced chromosomal damage.
Nobiletin, a flavonoid present in the peel of many citrus fruits, especially prevalent in C nobilis, in a mixture with ethanol and glycerol, applied to the ears of hamsters, once daily for 14 days, showed a decrease in the amount of triacylglycerol on the bskin surface, reduced the sizeof and amount of oil produced by the sebaceous glands. Triacylglycerol is the main constituent of mammalian fat and a major component of sebum. Results suggest a potential in the treatment of acne.

Operational Definition of Terms.

Alkaloids. These are chemical substances which are nitrogen heterocycles which occur mainly in plants as their salts of common carboxylic acids. They constitute an indispensable and most potent group of substances for the treatment and mitigation of functional disturbances and relief from suffering. Alkaloids are anti-hypertensive, antineoplastic agents and demonstrate anti-tumor activity.

Anthraquinones. An aromatic organic compound derivative of anthracene with a formula of C14H8O2. They naturally occur in plants, fungi, lichens and insects where it serves as a basic skeleton for their pigments. Anthraquinone derivatives tend to have laxative effects.

Cardiac glycosides. A chemical group of glycosides used to treat heart failure and irregular heart beat . An example is digitoxin.

Extract. This refers to the solution obtained from the leaves of mandarin orange peel.

Flavonoids. A naturally occurring phenolic compound belonging to a large group that includes many plant pigments. Flavonoids have beneficial effects in the human diet as antioxidants, neutralizing free radicals which damage body tissues and leads to heart disease, stroke and cancer.

Phytochemical Screening. It is a series of tests that determines the presence or absence of certain chemical substances present in a plant. The chemical considered in this study are reducing sugars, anthraquinones, terpenoids, flavonoids, saponins, tannins, alkaloids, and cardiac glycosides.

Reducing sugar. A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone group.

Saponins. These are glycosides with distinctive foaming characteristics. They consists of a polycyclic glycone that is either a choline steroid or triterpenoid. Attached via C3 and ether bound to a sugar side-chain.

Tannins. They are substances which occur as mixture of polyphenols which are very difficult to separate since they do not crystallize.
Terpenoids. The terpenoids, sometimes called isoprenoids, are a large and diverse class of naturally-occurring organic chemicals similar to terpenes, derived from five-carbon isoprene units assembled and modified in thousands of ways. Most are multicyclic structures that differ from one another not only in functional groups but also in their basic carbon skeletons. These lipids can be found in all classes of living things, and are the largest group of natural products.
Plant terpenoids are used extensively for their aromatic qualities. They play a role in traditional herbal remedies and are under investigation for antibacterial, antineoplastic, and other pharmaceutical functions.
Methodology

This section presents the design of the study, materials and experimental procedures and the statistical treatment of data.

Design of the Study
This study made use of the experimental research design in actual laboratory set- up. Two phases were included in the pursuit of this study:
Phase 1: Preparation of plant samples and extraction using distilled water and ethyl alcohol.
Phase 2: Phytochemical screening was done to determine the presence of reducing sugars, anthraquinones, terpenoids, flavonoids, saponins, tannins, alkaloids, and cardiac glycosides in the peel extract

Procedures:

Collection and Extraction of Plant Materials

The fruits of C. Nobilis were peeled off, the peel blanched with denatured alcohol to stop any enzymatic activity that will make the peels rot, then the peels were air dried for a week. After air drying, the plant samples were turned into uniform powder using a blender, and then weighed. Eighty-five grams of the powder was obtained, after which 850 mL denatured alcohol was added to this sample to extract the secondary metabolites present. This set-up was filtered after 48 hours using Whattman No. 1. The extracts were concentrated using a water bath.

Phytochemical screening

Phytochemical screening were performed using standard procedures.

Test for reducing sugars (Fehling’s Test)
The aqueous extract (0.5g in 5 mL water) was added to boiling Fehling’s solution (A and B) in a test tube. The solution was observed for a color reaction.

Test for anthraquinones
0.5 g of the extract was boiled with 10 mL dilute Sulfuric acid and filtered while hot. The filtrate was shaken with 5 mL of chloroform. The chloroform layer was pipette into another test tube and 1 mL of dilute ammonia was added. The resulting solution was observed for color changes.

Test for terpenoids (Salkowski Test)
To 0.5 g of the extract was added 2 ml Chloroform. Concentrated sulphuric acid at 3 mL was carefully added to form a layer. A reddish brown coloration at the interference indicates the presence of terpenoids.

Test for flavonoids
Dilute ammonia (5mL) was added to a portion of an aqueous filtrate of the extract. Concentrated sulphuric acid (1mL) was added. A yellow c9oloration that disappear on standing indicates the presence of flavonoids.

Test for saponins
To 0.5 g of extract was added 5 ml distilled water in a test tube. The solution was shaken vigorously, and observed for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously after which it was observed for the formation of an emulsion.

Test for tannins
About 0.5 g of the extract was boiled in 10 mL of water in a test tube and then filtered. A few drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black coloration

Test for alkaloids
0.5 g of extract was diluted to 10 mL with acid alcohol, boiled and filtered. To 5 mL of the filtrate of the filtrate was added 2 mL of dilute ammonia. 5 mL of chloroform was added and shaken gently to extract the alkaloidal base. The chloroform layer was extracted with 10 mL of acetic acid. This was divided into two portions. Mayer’s reagent was added to one portion and Draggendorff’s reagent to the other. The formation of a cream (with Mayer’s reagent) or reddish brown precipitate (with Draggendorff’s reagent) was regarded as positive for the presence of alkaloids.

Test for cardiac glycosides (Keller-Killiani Test)
To 0.5 g of extract diluted to 5 mL in water was added to 2 mL glacial acetic acid containing one drop of ferric chloride solution. This was underplayed with 1 mL of concentrated sulphuric acid. A brown ring at the interface indicated the presence of deoxysugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just above the brown ring and gradually spread throughout this layer.
Results and Discussions

Results on the phytochemical screening of the Citrus nobilis peel are presented in the following table.

Tests Citrus nobilis peel Results Indication Reducing sugars Formation of brown precipitate Presence of reducing sugar Anthraquinones No reaction Absence of Anthraquinones Terpenoids (Salkowski test) Brown coloration at the interface Presence of terpenoids Flavonoids A yellow coloration Presence of flavonoids Saponins (froth test) No froth formed Absence of saponins Tannins Bluish black coloration Presence hydrolysable tannins Alkaloids (Draggendorff’s test) Reddish brown precipitate Presence of alkaloids Cardiac glycosides (Keller-Killiani Test) Brown ring observed at the interface with a violet ring below Presence of cardiac glycosides
Reducing sugars
Reducing sugars are present in the peel, as observed in the Fehling’s test.

Anthraquinones
Anthraquinones were found to be absent in the peel of dalanghita.

Terpenoids.
Terpenoids were present in the peel. Terpenoids are under investigation for antibacterial, antineoplastic, and other pharmaceutical functions.
Flavonoids
Flavonoids can also be found in the peel of dalanghita as evidenced by the formation of a red color using Wilstatter test.
Flavonoids have antiviral, anti-inflammatory and cytotoxic properties (Capal, 1992)

Saponins
The peel of C. nobilis do not contain saponins. On Froth test, the peel extract did not form froth that persisted for 30 minutes.

Tannins
Using Ferric chloride test, the presence of tannins was detected by the formation of a brownish-green precipitate.
Tannins are possible sources of chemicals for the treatment of diarrhea and extensive burns and maybe used rectally for the relief of various rectal disorders (Santos, 1985). It is also used n the treatment of bed sore and weeping ulcers. It was formerly used for sore throat and stomatitis.

Alkaloids. As gleaned from the summary of results. Dalanghita peel yielded positive result fo alkaloids using Dragendorff’s test. This is evidenced by the formation of reddish brown precipitate.
Alkaloids are widely used in medicines like morphine, codeine, etc. Alkaloids are anti-hypertensive, antineoplastic agents and demonstrate encolytic property. It is used to relieve nasal congestion, stop hemorrhage, combat malaria and dilate the pupil of the eye and also used as a muscle stimulant. (The US Educator Encyclopedia, 1984).

Cardiac Glycosides
The peel extract of C. nobilis contains cardiac glycosides. This was evidenced by the formation of a reddish brown color at the interface using the Keller -Kiliani Test.
Cardiac glycosides have effects on the heart and kidneys and affect the contractions of the heart muscles.

Conclusions

The dalanghita (Citrus nobilis) peel extracts contain reducing sugars, terpenoids, alkaloids, cardiac glycosides, flavonoids and tannins. These substances can become excellent sources of therapeutic substances and as additive to cosmetic products.

Recommendations

1. A follow up study should be conducted to quantify, isolate and identify the type of reducing sugars, terpenoids, alkaloids, cardiac glycosides, flavonoids and tannins present in the peel of dalanghita.
2. Tests on the presence of other compounds are highly recommended, to further explore the potential of dalanghita peel as a source of alternative drug and other industrial applications.
3. Pharmacological testing should be done.
4. Other parts of dalanghita could be used for phytochemical screening and bioassays.
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C. madurrensis Mandarin orange (Engl.) C. nobilis Naranjita (Span.) C. papillaris Ransas (Bik.) C. reticulata Sintonis (Tag.) C. webberi Sinturis (Tag.) Tison (Tag.) Tangerine orange (Engl.) Tangor (Engl.) Ch’en P’i (Chin.)