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CHAPTER 1 INTRODUCTION 1.

1 GENERAL INTRODUCTION

Herbs known as ulam can be defined as part of the vegetation consists of shoots, leaves, stems, cabbage, beans, fruits, tubers and flowers are eaten raw, blanched, steamed or boiled before eating. Herbs famous as the main daily meal in the Malay community in the village and nowadays available in the area from the city as well. As a side dish eaten directly without cooked, such mineral content of minerals and vitamins such as calcium (Ca, phosphorus (P), iron (Fe), sodium (Na) and potassium (K) in a herbs does not destroyed by heat . For example, the scientific name of herb that is kaduk (piper

sarmentosum) had calcium (Ca), phosphorus (P), and iron (Fe) was the highest compared with other herbs. Other than that, herbs also provide fiber that is really needed in the human digestive system. Fiber plays a role as a filler on waste and facilitate the movement of stool in the intestinal tract (J. Trop. Agric. and Fd. Sc. 39(1)(2011). In the chemical structure of herbs, there are some important components such as phenol, flavonoids, tannins and saponins. The components role as to provide color and nutrients on herbs. According to the theory, flavonoids and phenol components connected closely in antioxidant activity.

This experiment was applied to the qualitative and quantitative analysis in determination of phytochemicals constituents, total phenol content (TPC) and antioxidant activity (DPPH). The qualitative is very essential for identifying the compound present in the herbs (C. Chitravadivu, 2009). In addition, phytochemicals is chemical compounds that occur naturally in plants that are responsible organoleptic properties such as color and smell. The phytochemical screening of the plants revealed the presence of flavonoids, saponins, tannins and phenol for herbal extraction (Tropical Journal of Pharmaceutical Research, September 2008) . Flavonoid play a variety of biological activities in plant. Its are the most important plant pigments for flower coloration producing yellow or red/blue pigmentation in petals designed to attract pollinator (Asian J. Plant Sci. Res., 2012). Tannin is defined accuring polyphenols compound of high enough molecular weight to form complexes proteins. Phenol is natural product but most phenolic have two or more hydroxyl group and phenol is the largest contributor of antioxidation production in plant meanwhile saponins are glycoside compounds often referred to as a natural detergent because of their foamy texture and its include antioxidation effects (Pak. J. Pharm. Sci., Vol.22, No.3, July 2009).

1.2

PROBLEM STATEMENT

Most of Malaysian societies does not know detail about the nutrients content in Malaysian herbs. According to the tradisional believes, Malaysian herbs contain many of benefits such as can prevent the chronic disease, slow the aging process and kept health care interior but there is no detail research about the nutrient content presence in Malaysian herbs nowadays. In addition, not all of Malaysian herbs are rich in phenol content and antioxidant completely. So on this research is carried out and focuses on ; 1. Does Malaysian herbs contain phytochemicals? 2. Which among the herbs contain highest phenol content? 3. Does the phenol influent the antioxidant activity?

1.3

OBJECTIVES
The objectives on this research is ; 1. Determine the qualitative phytochemicals in 9 different types of Malaysian herbs. 2. Determine the total phenol content (TPC) in 3 different selected herbs by using Folin- ciocalteou method. 3. Determine the antioxidant activity using a DPPH method in 3 different selected herbs.

4. To the relationship between total phenol content (TPC) and antioxidant activity among the 3 different type of Malaysian herbs.

1.4

SCOPE ANALYSIS

This experiments were done to determine the qualitative phytochemicals content which is flavonoids, saponins, tannins and phenol in 9 different types of herbs analysis to selected. 3 Types of herb were tested quantitatively of total phenol content (TPC) and antioxidant activity by using a Follin-Ciocalteau (TPC) and DPPH scavenging activity (antioxidant activity). Thus, the 3 different types of sample were analyzed quantitatively at the end of experiment to investigate the highest antioxidant and also relationship between TPC and antioxidant activity among the 3 different types of herb.

APPENDIX
APPENDIX 1 : FLOW CHART DRYING OF PLANT MATERIAL (HERBS)

The herbs was selected and resolved one by one into the tray

The herbs were dried in the oven drying in 24 hours at temperature 60c

The dried herbs was put into the container as a dry stock for further next uses

APPENDIX 2 : PREPARATION OF SAMPLES EXTRACTION OF DCM METHOD (DI-CHLORO METHYL)

5g of dried finely powdered herbs was taken in a beaker and 200ml of Dichloromethane(DCM) was added

The mixture was heated on a water bath at 39.6C for 6 hours

Then the DCM extract was filtered using filter paper (30 to 50 M) with assist of vacuum pump, respectively and then the residues were re-extracted use solvent Methanol for next extraction

The liquid extract of DCM was place in beaker and closed with aluminium foil to avoid from defect and leave the DCM extracts until it fully volatile

APPENDIX 3 : PREPARATION OF SAMPLES EXTRACTION OF METHANOL METHOD

The filtrate of herbs DCM extract was taken in a beaker and 200ml of Methanol (MeOH) was added

The mixture was heated on a water bath at 64.7C for 6 hours

Then the Methanol extract was filtered using filter paper (30 to 50 M) with assist of vacuum pump, respectively

The liquid extract of Methanol was place in beaker and closed with aluminium foil to avoid from defect and leave the Methanol extracts until it fully volatile

APPENDIX 4 : FLOW CHART OF FLAVANOID TEST (PHYTOCHEMICALS TEST)

BY USING NAOH TEST 5 ml of 1% hydrocholoric acid (HCL) are extract were shaken with sodium hydroxide

Yellow colored were appears

Observe the result

APPENDIX 5 : FLOW CHART OF TANNIN TEST (PHYTOCHEMICAL TEST )

BY USING BAYMERS TEST 1 drop of Ferric Chloride was added to 2 ml of the extract

The bluish or greenish black coloration indicates the presence of cathecol tannins

Observe the result

APPENDIX 6 : FLOW CHART OF SAPONIN TEST (PHYTOCHEMICALS TEST)

2 ml of extract added with 2 ml of water and shake well

Observe the result

APPENDIX 7 : FLOW CHART OF PHENOL TEST (PHYTOCHEMICALS TEST)

Herbs extract was mixed with 2 ml of 20% solution of Ferric Choloide

Blue-green or black coloration will indicates the presence of phenols

Observe the result

APPENDIX 8 : FLOW CHART OF TOTAL PHENOL CONTENT TEST (TPC) (FOLIN-CIOCALTEAU METHOD)

5 ml of Folin-Ciocalteu reagent (10%, v/v) was mixed with 4 ml 5% (w/v)Na2CO3 solution, and then 0.5 ml (1 mg/ml) sample solution was added

15 minutes incubation at room temperature in dark, the absorbance was measured at 765 nm

The results were presented in (mg) Gallic acid equivalent ( mg GAE/g) plant extract

Repeat the experiment using Gallic acid as standard

APPENDIX 9 : FLOW CHART OF ANTIOXIDANT ACTIVITY TEST ( DPPH METHOD)

2.0 ml of selection herbs extract at difference concentrations (5,10,15,20 mg/ml), 6 ml of methanol standards and 2.0 ml of DPPH dehydrated methanol solution separately

After 30 minutes incubation at room temperature in the dark, the absorbance was measured at 517 nm.

Observe the result

APPENDIX: IMAGE OF REAGENTS

Ferric chloride 2%

Ferric Chloride 10%

Hydrochloric acid (HCL) 1%

Natrium hydroxide (NAOH)10%

APPENDIX : IMAGE OF PHYTOCHEMICAL TEST

Phenol test for Methanol ( pucuk ubi kayu)

Phenol test for DCM Extraction ( Ulam raja)

APPENDIX : IMAGE OF SOLVENT