An In vitro Study of Orthosiphon stamineus (Misai Kucing) Standardized Water Extract as a Chemolytic Agent in Urolithiasis.

Background: Orthosiphon stamineus was reported to have diuretic effects in experimental rats, and this leads to inhibition of kidney stones through the abundant levels of minerals and flavonoids in it. This study aimed to determine the in vitro effects of O. stamineus water extract as a potential chemolytic agent in urolithiasis. Materials and
Methods: In this prospective experiment, a total of 15 stone samples collected from patients who underwent stone extraction were used in each concentration (4 mg/ml, 2 mg/ml, and 1 mg/ml) of the O. stamineus extract and control solution. The effects of pH change in the chemolysis of the stones were assessed using the O. stamineus extract 4 mg/ml under pH 7 and 8.
Results: The percentage weight reduction of calcium oxalate stone was highest in the 4 mg/ml concentration. O. stamineus extract 4 mg/ml showed a better effect in terms of chemolytic action on calcium oxalate stone than the potassium citrate solution (70% vs. 41%). Regarding the calcium oxalate stone, the percentage weight reduction has shown about 70% in the pH 5, 48% in pH 7, and <10% in pH 8. The percentage weight reduction of uric acid stone was determined as 47%, 11%, and 14% for pH 5, 7, and 8, respectively. The percentage weight reduction of combination stone was 40%, 60%, and 80% in the pH 5, pH 7, and pH 8, respectively. Data analysis showed that the percentage weight reduction of combination stone was significantly different between acidic, neutral, and alkaline conditions (P = 0.027). Conclusions: In this in vitro study, we are able to show that O. stamineus water extract do have some dissolving capability of urinary stones. Copyright:

INTRODUCTION

Urolithiasis (also known as kidney stone or urinary calculi) has blighted human antiquity, with historical background dated as far back as ancient time. The prevalence of urolithiasis was 4%–8% worldwide,[1] with the United States having the highest percentage of 8% in male population.[2] The incidence of kidney stone in Malaysia as reported between 1962 and 1976 by Sreenevasan in 1981, at an average of 221 cases annually.[3] A recent study has shown that a prevalence of urinary stone in Kelantan state, Malaysia, was 3.9%–3.7% in 100,000 population.[4]

The main scope of medical therapy in urolithiasis is multidimensional approach to modify some of the risk factors involving increase in daily fluid intake, limiting sodium intake, calcium and oxalate intake, as well as reduction in consumption of animal protein. Some of drugs used include potassium citrate and allopurinol, while infectious stones are treated with stone removal and prophylactic antibiotics.[5] Medical expulsion therapy with either alpha antagonist such as tramsolusin, terazosin, or calcium channel blockers such as nifedipine was shown in a systematic review of medical expulsion therapy that it enhances the stone expulsion rate compared with standard therapy for moderately sized distal ureteral stone.[67] Despite the advancement in the management of patients with stone disease, with the introduction of endourological procedure avoiding the use of open surgery, it is not without complications.[8910]

Herbal medicines were being used along with modern medicine in many countries. Misai Kucing (Orthosiphon stamineus) is a medicinal herb found mainly throughout Southeast Asia and tropical Australia. The plant is an herbaceous shrub, with a unique flower. The flowers have fine filaments that tally cat's whiskers hence its Malay name, Misai Kucing (Cat's whiskers). It was discovered that it contained terpenoids (diterpenes and triterpenes), polyphenol (lipophilic flavonoids and phenolic acids), and sterols.[11] There are three main polymethoxylated compounds in Misai Kucing, namely sinensetin, eupatorin, 3'-hydroxy-5,6 74 tetramethoxyflavone, and rosmarinic acid.[1112] In Malaysia, Misai Kucing tea was produced and used in the treatment of bladder inflammation, gout, and diabetes.[13]

The O. stamineus was reported to have diuretic effects in experimental rats, and this leads to inhibition of kidney stones through the abundant levels of minerals and flavonoids in it.[14] These compounds were known chemical inhibitors preventing the growth and aggregation of crystals.[15] In view of the above findings, we thought that there may be direct action of the O. stamineus to already formed stones as a chemolytic agent.[14] Our experimental study was carried out to assess the in vitro effects of O. stamineus (Misai Kucing) water extract as a potential chemolytic agents in urolithiasis.

MATERIALS AND METHODS

Plant materials and chemicals

The standardized water extract of O. stamineus was obtained from the Institute for Medical Research, Kuala Lumpur, Malaysia. The extract was kept in an airtight container until further experimentations. The extract was analyzed using Agilent Technologies Series 1100 HPLC System (Agilent Technologies, Polo Alto, CA, USA). Based on the analysis, it was reported to contain rosmarinic acid 8.99%, sinensetin 0.35%, eupatorin 0.45%, and 3-hydroxy-5,6 74 tetramethoxyflavone 0.3%.[16] Three concentrations of the plant extract tested for their chemolytic activity were 4 mg/ml, 2 mg/ml, and 1 mg/ml, which were prepared at the time of experiment.

Kidney stone specimen

Kidney stone samples were collected from patients who underwent stone extraction by either open surgery or endourology procedures at the Hospital Universiti Sains Malaysia (USM), Kelantan, Malaysia. The stone samples (sizes range 0.1–2.0 g) used in the experiment were kept in sterile bottles container for a maximum time of 6 months with an average ranged of 1–3 months.

Chemical analysis of the stone samples was done at the School of Health Sciences Laboratory, USM, Kelantan, Malaysia. Fourier transform infrared (FTIR) spectrometer Bruker Tensor 27 (Bruker Optik GmbH, Ettlingen, Germany) was used to identify the different stone types.

Experimental design

This is a prospective in vitro experiment to assess the effect of O. stamineus (Misai Kucing) as a chemolytic agent on kidney stones for a period of 8 weeks.

The kidney stone samples were grouped according to their chemical composition (i.e., calcium oxalate stones, uric acid stones, and calcium oxalate and uric acid stones). In each group, the dissolution power of O. stamineus extracts was assessed under three different concentrations of 4 mg/ml, 2 mg/ml, and 1 mg/ml under acidic pH 5 with potassium citrate as a positive control, while Hartmann's solution as a negative control. Hartmann's solution contains sodium chloride, potassium chloride, calcium chloride dehydrate, and sodium lactate 60%. Each liter of solution contains 6 g sodium chloride, 0.4 g potassium chloride, 0.27 g calcium chloride dehydrate, 5.16 g sodium lactate 60%, and water for injections. The pH range is 5.0–7.0.

A total of 15 small stones were used in each stone grouping; three stones per set were used (n = 3) in each concentration of the O. stamineus extract and control solution. Each stone was immersed in the plant extract and control solutions in a separate sterile leak proof container. The volume of solution used for the immersion of the stone was determined based on the stone weight as 6 ml/100 mg of the stone. The solution was changed daily in both experimental and control groups. All experiments were performed at 37°C. A weekly weight reduction was recorded for 8 weeks.

The above procedure was done in all three types of stone groups: calcium oxalate, uric acid, and combination stone. At the end of the study, the percentage weight reduction of each stone was calculated using the formula below:

% stone weigh lost = [(initial weight − final weight)/initial weight] ×100

The concentration of the O. stamineus extract with the highest percentage dissolution was identified in the 4 mg/ml in all the stone groups.

The effects of pH change in the chemolysis of the three stone types were assessed using the O. stamineus extract 4 mg/ml under pH 7 and 8 for another 8 weeks. The percentage weight reduction of each stone was calculated. Results of this basic condition were compared with the acidic condition under pH 5. Hydrochloric acid 37% was used as acidifying agent, while potassium hydroxide 85% and sodium hydroxide were used as basic media.

The morphological pictures of the stones were taken using high-power microscope both pre-and post-experiments to determine changes in the stone configurations.

Ethical considerations

An ethical clearance was obtained from the Human Research Ethics Committee, USM, Kelantan, Malaysia (Reference Number: USMKK/PPP/JEPeM [252.4.(1.2)].

Statistical analysis

The percentage reduction in stone weight was determined. The results were evaluated using nonparametric, the Kruskal–Wallis test, followed by the Mann–Whitney U-test to identify differences between groups. Statistical analysis was carried out using SPSS/Win software (Version 22, SPSS, Inc., Chicago, IL, USA). The limit of significance was set at 0.05.

RESULTS

Morphological changes in stone physical structure

The morphological pictures of the stones taken using high-power microscope both pre- and postexperiments are shown in Figure 1.

Figure 1

High-power microscopic pictures of calcium oxalate stone, uric acid stone, and combination of calcium oxalate and uric acid stone. (a) Calcium oxalate stone pictures taken pre- and post-experiments; pre-experiment picture shows hard stone brown in color which grow in radial fashion from the nidus with rounding wedge at extremity, and post-experiment picture shows disappearance of irregular surface probably due to dissolution exposing the interior table layer in a well-arranged pattern. (b) Uric acid stone pictures taken pre- and post-experiments; preexperiment picture shows dark orange stone composes of small spherical region, and postexperiment picture shows stone with smooth interior surface. (c) Calcium oxalate and uric acid stone pictures taken pre- and post-experiments; pre-experiment picture shows yellow cluster of platelets, and post-experiment picture shows nodular surfaces and discoloration of the stone

Percentage reduction in stone weight

Table 1 shows the percentage mean weight reduction of each stone group at the end of 8 weeks under acidic pH 5. The concentration of the O. stamineus extract with the highest percentage weight reduction was identified in the 4 mg/ml in all the stone groups.

Table 1

The effects of Orthosiphon stamineus extract, Hartmann’s solution and potassium citrate on calcium oxalate stone, uric acid stone, and combination of calcium oxalate and uric acid stone under pH 5

	Type of stone
	Calcium oxalate	Uric acid	Combination*
Orthosiphon stamineus extract
4 mg/ml	69.47	47.03	42.43
2 mg/ml	41.52	23.61	29.34
1 mg/ml	63.39	44.04	14.15
Hartmann’s solution	30.19	44.34	37.01
Potassium citrate	41.20	54.66	52.86

*Calcium oxalate and uric acid stone. Data are percentage mean weight reduction of the stones for 8 weeks

However, no significant differences were observed in the percentage weight reduction of calcium oxalate stone, uric acid stone, and combination of calcium oxalate and uric acid stone between O. stamineus extracts of concentration 4 mg/ml, 2 mg/ml, and 1 mg/ml, Hartmann's solution, and potassium citrate when subjected to Kruskal–Wallis test (P > 0.05).

Pattern of reduction in stone weight under pH 5, pH 7, and pH 8

The effects of pH change in the chemolysis of calcium oxalate stone, uric acid stone, and combination of calcium oxalate and uric acid stone in O. stamineus extract concentrations of 4 mg/ml are shown in Figure 2. Data analysis showed that the percentage weight reduction of combination stone was significantly different between acidic (pH 5), neutral (pH 7), and alkaline (pH 8) conditions (P = 0.027). However, no significant differences were observed in the percentage weight reduction of calcium oxalate stone and uric acid stone between different pH when subjected to Kruskal–Wallis test (P > 0.05).

Figure 2

Pattern of reduction in weight of calcium oxalate stone, uric acid stone, and combination of calcium oxalate and uric acid stone under pH 5, pH 7, and pH 8 in Orthosiphon stamineus extract concentration of 4 mg/ml. (a) Calcium oxalate stone; (b) uric acid stone; and (c) calcium oxalate and uric acid stone. The percentage weight reduction of combination stone (i.e., calcium oxalate and uric acid stone) was significantly different between acidic (pH 5), neutral (pH 7), and alkaline (pH 8) conditions (P = 0.027). The percentage weight reduction of calcium oxalate stone and uric acid stone was not significantly different between different pH

DISCUSSION

Herbal medicines were being used along with modern medicine in many countries; however, many research was still required to demonstrate their efficacy in modern medicine. It was reported that more than 80% of developing countries' population rely on herbal or traditional medication as their primary intervention perhaps due to either higher cost or the apprehension on the side effects of modern medicine.[17] There are many claims both from traditional medical practitioners and part of community on the effects of O. stamineus as a cure in kidney stone disease. In the current study, we showed that there was a tendency for O. stamineus extract to have some dissolving capability of urinary stones.

Results of morphological pictures of the stones showed significant changes in physical appearance of stones. Preexperimentally, calcium oxalate stone was shown as hard stone brown in color which grows in radial fashion from the nidus with rounding wedge at extremity. However, in the postexperimental microscopic picture, it showed disappearance of irregular surface probably due to dissolution exposing the interior table layer in a well-arranged pattern. If the exposure was prolonged, we may assume that this inner table would be thinner due to erosion. The uric acid stone microscopic picture shows dark orange. It is composed of small spherical region in the preexperiment as compared with postexperimental microscopic picture with smooth interior surface. The stone has small needles that radiate from the center. The mixed stone, on the other hand, shows yellow cluster of platelets, these platelets are sharp and arranged in various orientations preexperimentally, whereas the postdissolution shows some nodular surfaces possibly due to chemolytic action on stone surface and discoloration of the stone.

The percentage weight reduction of calcium oxalate stone was highest in the 4 mg/ml concentration. O. stamineus extract 4 mg/ml showed a better effect in terms of chemolytic action on calcium oxalate stone than the potassium citrate solution (70% vs. 41%). These results showed that there are tendency and possibility of the extract to have some dissolving capability of urinary stones; however, the exact mechanism of action and the main components responsible for this effect require more investigation.[1819] This effect was reported by Atodariya et al.[20] after comparing the percentage dissolution of many stones by Dolichos biflorus seeds with cystone in oxalate stone which showed close similarity of stone reduction. They concluded that the effects were due to the presence of phenolic compounds, flavonoids, and steroids in that extract. Likewise, the same composition was reported in the O. stamineus.[1216] Furthermore, another report by Quazi Majaz et al.[21] showed a diuretic and some chemolytic action of Kalanchoe pinnatum plant on Wistar rats in both oral and intravenous routes which were also attributed to phenolic compounds and flavonoids. Medicinal plants with antioxidative properties such as Cynodon dactylon extract reduced the stone incidents, at least in part through increasing the total antioxidant capacity.[18] Metformin has been reported to suppress urinary crystal deposit formation through renal tubular cell protection and antioxidative effects.[22] Consistent with this observation, EtOAc extract of O. stamineus Benth. has shown inhibition of the stones formation by improving oxidative stress and inflammation mediated by glycerophospholipid metabolism.[14]

The study of uric acid stone showed that potassium citrate has higher dissolution capacity compared with the O. stamineus extracts. This effect of potassium citrate on stones was reported by many researchers, and it has been considered as a standard mode of treatment in urology for uric acid stones.[232425] Even though the percentage reduction observed was less and statistical analysis was not significant, there is still possibility for the extract to have a preventive role in uric acid stone where it was reported to cause diuretic effect in Wistar rats which in turns prevent the micro-aggregate of stone particles, leading to stone formation.[112627]

In the combination stone study, O. stamineus extracts show that the greater percentage reduction was observed in the 4 mg/ml followed by 2 mg/ml and the least was seen in 1 mg/ml concentration. However, the percentage reduction seen in the potassium citrate was highest in comparison to the O. stamineus extracts, hence confirming its effectiveness as a chemolysis agent. However, the differences were not statistically significant. In this study, the possible reason of nonsignificant difference could be from small sample size or presence of other stone component which was not identified by FTIR spectroscopy, and finally natural stones were used in the study as oppose the artificial stones which were commonly used.

In the second phase of this study, we were looking for the effects of pH changes on the chemolytic ability of O. stamineus. In the first phase of this study, results consistently showed that the best stone dissolution were obtained by using extract at concentration of 4 mg/ml. Hence, this concentration was chosen to test whether there is any additional effect when the pH was changed from acidic to alkaline. Regarding the calcium oxalate stone, the overall percentage reduction has shown about 70% in the acid medium (pH 5), 48% in neutral (pH 7), and <10% in the alkaline condition (pH 8). It can be postulated that acidic medium is a better environment for chemolysis of calcium oxalate stone. In the pH 8, there was a static weight change of calcium oxalate stone in the initial four weeks, then a sudden dissolution and it plateau again. The dissolution power of O. stamineus 4 mg/ml was masked by holding power of the alkaline solution which prevents chemolysis to occur. In the neutral environment, there was little inhibition; hence, there was slow but steady reduction as compared with the alkaline solution. In the pH 5, however, the dissolution was slow at initial stage but rapidly increases up to the 8 weeks, which implies that calcium oxalate stone requires acidic medium for effective treatment as reported.[28]

The uric acid stone was also assessed under the same experimental conditions. The percentage weight reduction was determined as 47%, 11%, and 14% for pH 5, 7, and 8, respectively. These results have shown that pH 5 was quadruple effective when compared to neutral condition. In the pH 5, there was a steady initial decrease up to 4th week and rapid reduction in the consecutive 2 weeks then became static again. In the neutral condition, there was slow reduction throughout the period. However, in the alkaline environment, it showed static reduction up to 8th weeks and then ascend at the 7th–8th week. This difference in the pattern of reduction could be due to other unidentified component of the stone or can be considered as an experimental outlier.

Exposure of uric acid stone to alkaline medium results in dissolution in most of the cases as reported by Honda et al.[24] They reported a retrospective study achieving 73% in chemolysis and concluded that it is effective in uric acid treatment. However, in our study, there was little chemolysis observed in the alkaline solution. This could probably be due to the presence of another chemical component which counteracts the alkaline effect. This phenomenon was reported by Funahashi et al.[28] where they reported unsuccessful uric acid chemolysis due to the presence of mixture of calcium oxalate and phosphate combined with inability to alkalinized the solution. Therefore, it can be deducted that possible presence of other stone component has possibly led to lower percentage dissolution.

The overall percentage weight reduction of combination stone was 40%, 60%, and 80% in the pH 5, pH 7, and alkaline pH of 8. In the pH 5, there was some chemolysis at the initial 4th week; then, surprisingly, the stone size increases which could be due to wrong data collection. However, in the pH 7 and 8, there were stable weight reductions throughout the experimental period. From the result, it can be seen that the test solution has highest chemolytic action in the pH of 8 followed by pH 7 and the least goes to acidic medium. Even though there was no direct report which explains this action, there was a study which correlate the influence of pH and chemolysis in uric acid stone.[232429] They were able to show significant chemolysis of uric acid stone when it is treated with alkaline solution. This also supports the earlier postulation regarding the presence of other chemical composition affecting the chemolytic action. In the acidic medium, the results of weight reduction were less compared with the alkaline possibly due to calcium oxalate presence as reported by Funahashi et al.[28]

From the results of the second phase of this study, there is high possibility that the O. stamineus 4 mg/ml could be an alternative option in the treatment of uric acid stone and combination stone. However, it should be noted that in all the experiments, statistical analysis was done using nonparametric, the Kruskal–Wallis test which showed no significant differences in percentage reduction in weight of stones except in the combination stone.

This study has limitations. First, the experiments were done in vitro studies, so metabolism of the O. stamineus water extract, pharmacokinetics, and pharmacodynamics parameters were not considered. Hence, further study is required to assess the in vivo effects of Orthosiphon staminues water extract as a potential chemolytic agents in animal and human volunteers before proceeding with clinical trial. Future study should be performed with higher number of samples, similar stones sizes, and other types of O. stamineus extract such as ethanol extract. This will minimize the problem of inadequate samples which may lead to insignificant results post statistical analysis.

CONCLUSIONS

This study provides preliminary scientific evidence about the tendency for the O. stamineus water extract to have a prophylactic potential in terms of preventing kidney stones formation due to its chemolytic action. Based on the results and literature information regarding the composition of this plant, we inferred that O. stamineus extract has potential application in the prevention of stones recurrence in humans. However, the exact mechanism of this protective effect is not completely understood. Chemolysis of different renal stones under different in vitro experimental conditions may be ascribed to its phenolic and flavonoids compounds. On the basis of findings of the current study, the percentage reduction in weight of stones was seen, but the mechanism of action was not fully understood. Further study with larger number of samples is required to prove the ability of the extract to act as chemolytic agents.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.