Hemorrhoid Therapy with Medicinal Plants: Astringency and Inhibition of Lipid Peroxidation as Key Factors

MATERIALS AND METHODS

The study was carried out (May 2007 to January 2009) in our laboratory at the Department of Pharmacognosy, Faculty of Pharmacy, University of Lagos, Nigeria.

Sample Collection
The plant materials were earlier identified courtesy of Mr. Wale Ekundayo by comparing with herbarium specimens at FRIN (Forest Research Institute of Nigeria herbarium). Collection was through purchase from a local market in Lagos Nigeria, courtesy of Mr. Isaac Adeleke of the Department of Pharmacognosy, Faculty of Pharmacy, University of Lagos. The plant materials were dried in the oven at 50°C for 5 days, powdered, wrapped in newspaper and stored at room temperature until needed for the tests.

Extraction Procedure
A modified method of Muanya and Odukoya (2008) was used. Fifty grams of the powdered sample was weighed, put in a separating funnel and 300 mL of ethanol added. This was left to percolate for a week, the ethanol extract was filtered and the marc re extracted with 1x200 mL ethanol.

The ethanol extract was bulked together and filtered. The filtrate was concentrated by drying on a water bath by gentle heating. The dry extract was weighed, put in a sample container, properly labeled and kept in the refrigerator at 4°C until needed.

Source of Bovine Serum Albumin
Fatty acid free BSA (Sigma A-6003) was used.

Preparation of Buffer Solution
9.8 g of NaCl was dissolved in 800 mL of water, 11.5 mL glacial acetic acid was added, the solution was adjusted to pH 4.9 with a solution of NaOH and final volume made up to 1 L.

Qualitative DPPH Radical Scavenging Activity
Radical scavenging activity of plant extracts was determined by slightly modified method of Miliauskas et al. (2004) as used by Sofidiya et al. (2006). One milligram per milliliter ethanolic stock solution of extract was prepared and to this was added 0.5 mL ethanol solution of DPPH^. (1 mM). The mixture was shaken and then left to stand at room temperature for 10 min and colour reaction was noted. α-Tocopherol (Sigma) was used as standard antioxidant.

Determination of Astringency
A modified method of Hagerman and Butler (1978) was used. This involved the formation of a protein tannin complex. One milliliter extract was added to 2 mL solution of BSA (Bovine Serum Albumin 1 mg mL^-1 in buffer solution prepared above). This was mixed together and incubated for 24 h at 4°C in the refrigerator. The mixture was centrifuged at 1500 r for 12 min and the supernatant poured off. The precipitate was dissolved in 5 mL mixture of [1% Sodium Dodecyl Sulphate (SDS) and 5% v/v Triethanolamine (TEA) in distilled water]. One milliliter of acidic FeCl₃ was added, vortexed immediately and allowed to stand on the bench at room temperature for 10 min.

The tannin-BSA complexes present in the precipitate was measured spectrophotometrically (UV Spectrophotometer, Thermo Spectronic Genesys 20 model) using ferric chloride blue chromophore at an absorbance maximum of 510 nm and compared to a standard curve prepared with tannic acid. Astringency was reported as mg L^-1 tannic acid equivalents.

Determination of Total Phenol Content
Total phenol contents in the extract were determined by the modified Folin-Ciocalteu method of Wolfe et al. (2003) as used by Sofidiya et al. (2008). An aliquot of the extract was mixed with 5 mL Folin-Ciocalteu reagent (previously diluted with water 1:10 v/v) and 4 mL (75 g L^-1) of sodium carbonate.

The mixture was allowed to stand for 30 min at room temperature and the absorbance was measured at 760 nm spectrophotometrically. The final results were expressed as Gallic Acid Equivalents (GAE).

Determination of Total Flavonoid
Total flavonoids were estimated using the method of Ordonez et al. (2006). To 0.5 mL of sample, 0.5 mL of 2% AlCl₃ ethanol solution was added. The absorption was measured at 420 nm after 1 h 10 min, at room temperature. Total flavonoid contents were calculated as rutin from a calibration curve.

Lipid Peroxidation
Antioxidant efficacy of the extracts towards lipid peroxidation in raw and cooked fish homogenate of S. japonicum was measured by thiobarbituric acid reactivity method. Thiobarbituric acid (TBA) reactivity in the homogenate was determined by following a modified method of Luotola and Luotola (1985) as used by Muanya and Odukoya (2008) and expressed as MDA equivalent/mg of tissue. The TBA chromogen (intensity of the pink coloured complex) was measured at 532 nm against blanks using UV Spectrophotometer, Thermo spectronic (Genesys 20 model). Water-soluble vitamin E was used as reference standard. A graph of absorbance against concentration was plotted using the data obtained for the pure vitamin E. The TBARS of the extracts were evaluated from the standard curve (absorption against concentration of tetraethoxypropane) and expressed as MDA equivalent/mg of tissue.

Statistical Analysis
Three replicas were made for each experiment and all the data presented as the Mean±SEM. A regression analysis, using Excel for Windows Software, was established between astringency, lipid peroxidation, total phenols and flavonoid contents of different plant extracts.

RESULTS

Astringency value, total phenol and flavonoid content of plant extracts are presented in Table 1. Astringency of extracts was in the order of Spondias leaves>Dialium seeds>Dialium leaves> Newboldia leaves>Kigelia fruit>Spondias fruit>Kigelia bark>Harungana bark>Newboldia bark> Harungana leaves>Adansonia leaves>Achyranthes leaves. Astringency correlated positively with total phenols (R² = 0.7944), while correlation with flavonoid was low (R² = 0.059) this is due to the fact that the colour development in the Folin Ciocalteu procedure is due to the transfer of electrons at basic pH to reduce the phosphomolybdic/phosphotungstic acid complexes to form chromogenes thus measuring only phenols that will not bind to proteins. There was no correlation between total phenol and flavonoid content (R² = -0.0387).

Qualitative determination of free radical scavenging activity showed that all the of the extracts produced a positive colour change from purple to yellow.

Absorbance decreased with increasing concentration of plant extract and thus a resultant increase in lipid peroxidation as shown in Table 2 and 3. Malondialdehyde (MDA) is formed during oxidative degeneration as a product of free oxygen radicals, which is accepted as an indicator of lipid peroxidation (Draper, 1999).

Table 1:	Astringency value, total phenol and flavonoid content of plant extracts

Table 2:	TBARS values (mean of 3 readings ± SEM) with raw fish homogenate in plant extracts
ND = Not Done

Table 3:	TBARS values (mean of 3 readings ± SEM) with cooked fish homogenate in plant extracts
ND = Not Done

A rapid, simple and inexpensive method to measure antioxidant capacity involves the use of the free radical, 2, 2-Diphenyl-1-picrylhydrazyl (DPPH). DPPH is widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors and to evaluate antioxidant activity.

The qualitative analysis of the ethanolic extracts of the plants was carried out using the DPPH method. The odd electron in the DPPH free radical gives a strong absorption maximum at 517 nm and is purple in color. The color turned from purple to yellow as the molar absorptivity of the DPPH radical at 517 nm reduces from 9660 to 1640 when the odd electron of DPPH radical becomes paired with hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-H.

Astringency was measured as the amount of tannin precipitated by a standard protein Bovine Serum Albumin (BSA). Astringency is not a taste like sweetness, bitterness, sourness or saltiness but a feeling, of dryness as a result of increased friction caused by tannin, which reduces lubrication by precipitating proteins. The method of Hagerman and Butler (1978) used in this study involved formation of protein-tannin complex between the tannin containing extract and the protein, BSA.

Tannins have long been known to interact with proteins so assay used was devised to take advantage of this phenomenon. Ferric ion complexes with the tannins and forms a blue chromophore with an absorbance maximum at 510 nm. The blue product was determined spectrophotometrically.

Since, the BSA protein does not react with ferric chloride or interfere with the colour reaction, this analysis provided a convenient assay for phenols in the plant extracts as it only measured the phenols that could bind to BSA and precipitate along with the protein. The tannin phenol present is expressed relative to tannic acid.

The absence of correlation between total phenol and flavonoid content has also been found by (Meda et al., 2005; Moein et al., 2008) and could be explained by the presence of some other chemical groups other than proteins present in the plant extracts that can also react with the Folin Ciocalteu reagent.

Total flavonoid was calculated with reference to rutin. Though rutin helps those with capillary or venous fragility, easy bruising and a tendency of wounds not to heal well (Misra and Parshad, 2000). The low astringency correlation with flavonoid content indicated that the venous constriction in hemorrhoid therapy in this study might be as a result of astringency due to phenols that could bind to BSA and precipitate along with protein and not flavonoids. This result differs from previous studies as flavonoids are known to be effective for pain from injuries and bruises and possess the ability to protect, strengthen capillaries, aid circulation, lower cholesterol and reduce hemorrhoids (Misra and Parshad, 2000; Sandhu and Singh, 2004).

Inflammation associated with anal itching results in scratching, which further traumatizes these tissues. This trauma causes bleeding into the affected tissues, such that hemoglobin is released from the red blood cells. When the hemoglobin is exposed to the hydrogen peroxide generated from neutrophils and xanthine oxidase in inflamed tissues, there is hemoglobin degradation and consequent release of catalytic iron ions and toxic free heme which are capable of initiating or aggravating lipid peroxidation (Gough et al., 2000). Astringency correlated positively with inhibition of lipid peroxidation (R² = 0.6596 with raw homogenate and 0.9220 with cooked homogenate). The results of our study indicate that there was protection of lipid peroxidation as evidenced by the low level of TBARS values with increasing concentration of the extracts suggesting a decrease in lipid peroxidation.

Fatty acids Omega-3 is an important polyunsaturated fatty acid found in fish oils and have been shown to decrease lipid peroxidation (Erdogan et al., 2004). Activity was more in cooked than raw fish homogenate. The present study confirms previous studies that cooking alters the physicochemical nature of the membranes thereby, the solubility and site of action is favoured by having more access to the radical and thus better antioxidant activity and effective inhibition of the free radical damage (Seeman, 1990; Muanya and Odukoya, 2008). Thus the inhibition of lipid peroxidation by these plant extracts is indicative of anti hemorrhoid activity.

Hemorrhoids refer to a condition in which the veins around the anus or lower rectum are swollen and inflamed.In hemorrhoid there is prolapsed of the veins in the anal area as a result of inflammation. Therefore the treatment of this condition requires herbs that will strengthen the veins and reduce inflammation and bleeding.

The herbal treatment in hemorrhoid therapy involves use of astringents. Astringency is not a taste like sweetness, bitterness, sourness or saltiness. It is a feeling of dryness or roughness that is sensed by the tongue as a result of increased friction between the tongue and the surfaces inside the mouth. This is caused by tannin, which reduces lubrication by precipitating proteins. Astringents will therefore cause coagulation of proteins in the cells of the perianal skin or the lining of the anal canal. This action promotes dryness of the skin, which in turn helps relieve burning, itching and pain. These extracts have an astringent effect which helps heal mucous membranes and exposed tissue and bring about contraction and firm up tissue.

It is proposed that these astringent herbs accomplish anti hemorrhoid activity by causing coagulation of proteins in the cells of the peri anal skin or the lining of the anal canal. These extracts have an astringent effect which helps heal mucous membranes and exposed tissues. They plug up minute leaks and holes in the veins and capillaries, promote vein elasticity and act as vasoconstrictors in the peri anal area. Thus promote dryness of the skin, which in turn helps relieve burning, itching and pain associated with hemorrhoids.