Evaluating the thrombolytic property of Juniperus Sabina tincture using spectrophotometry

INTRODUCTION

Thrombolytic agents are medications that dissolve intravascular clots. Streptokinase (SK), urokinase and recombinant tissue plasminogen activator are some thrombolytics administered in acute life-threatening conditions such as ST-elevation myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis and acute limb ischemia, for dissolving thrombi; they substantially reduce the morbidity and mortality of these conditions. However, they are not always risk-free and are quite expensive.^[1,2] The thrombolytic agent SK has revolutionised the treatment of acute myocardial infarction when used within 6 h of symptom onset.

Some homoeopathic medicines of plant origin, such as Arnica Montana, Millefolium and Juniperus Sabina (More commonly referred as J. Sabina), have been used for treating haemostatic disorders. Although these remedies are frequently prescribed in homoeopathic practice, studies confirming their utility in haemostasis are limited.^[3,4]

Homoeopathic medicines have to be subjected to efficacy assessment to increase scientific literacy for their therapeutic use.^[5] Investigating the thrombolytic activity of homoeopathic medicinal products in acute life-threatening conditions during clinical trials can pose considerable risks to patients. Biological in vitro models provide informative data reliably and consider the components that lead to experimental bias.^[6] A study by Prasad et al. showed a clot lytic effect in vitro using SK as a positive control and water as a negative control. Their study design can be adopted to evaluate the thrombolytic properties of new or unknown drugs.^[2,7] Zamanlu et al. established a simple and cost-effective quantitative method for measuring the activity of thrombolytic agents with high precision and accuracy using in vitro models by studying the optical properties of haemoglobin (Hb) (λ_max = 405 nm) during thrombolysis using a spectrophotometer.^[2] Wang et al. as a part of their exploratory in vitro experiment induced thrombolysis with 5000 and 7500 IU doses of SK in citrated male CD1 mice. They customised the cuvettes by placing a nylon web at the neck and measured the absorbance of the released Hb at a wavelength of 580 nm during clot lysis for a period of 3 h.^[8]

J. Sabina (homoeopathic remedy name: Sabina) is a homoeopathic medicine known for its administration for blood disorders including menstrual ailments. Human pathogenetic trials and clinical proving have shown Sabina to dissolve clots.^[4] The mother tincture (Q) is the homoeopathic preparation from the crude plant extract. It carries a drug strength of 1/10.^[9] Mother tinctures contain pharmacologically active constituents that are responsible for the phytochemical action and the therapeutic effect of a drug at the physiological level. Since we aimed to explore the thrombolytic property of Sabina in vitro, the mother tincture preparation was used in this study.

MATERIALS AND METHODS

The novel procedure for assessing thrombolysis developed by Wang et al. was adopted for this study.^[8] After a few trial runs and adaptations, the experiment was conducted. Approximately 3 mL each of freshly drawn blood from six healthy volunteers was used after ensuring the absence of any clinical history pertinent to haemostatic disorders or oral contraceptive medications. They were subjected to a simple basic clotting screen consisting of bleeding time (BT) and clotting time (CT) tests. The volunteers with normal CT and BT were recruited for the study after providing written informed consent. Ethical approval from Father Muller Institution Ethics Committee was obtained before the study. After drawing blood, 0.3 mL was immediately added to four 15 mL centrifuge tubes. The centrifuge tubes were kept upright and undisturbed in a temperature-controlled room (22–25℃) for 10 min, allowing blood clots to form naturally. Then, the tubes were covered with parafilm and the clots were incubated for 45 min in a water bath at a temperature of 37°C. In accordance with the routinely followed washing procedure, the clots were gently washed 5 times with normal saline. The blood samples were centrifuged for 2 min each time at 2200 rpm during the washing procedure. After washing, the clots were pre-weighed and uniform-sized clots were chosen to include in the experiment. An ultraviolet-visible (UV-VIS) spectrophotometer (SYSTRONICS-119) was used to record the absorbance of Hb at five wavelengths 405, 416, 541, 576 and 580 nm, respectively. A mesh of 1200 microns was introduced over the upper segment of the cuvette to hold the clots.

RESULTS

Blood samples were collected from six healthy subjects with a mean age of 34.5 (26–38) years. The thrombolytic activity of SK, HA, T1 and T2 was compared by recording the absorbance of Hb at five wavelengths, namely, 405, 416, 541, 576 and 580 nm. Although the experiment was carried out for 3 h, the absorbance levels of SK plateaued out in 1 h and no rise in absorbance occurred in any of the remaining samples. Hence, only the findings of the first 1 h are presented here for the sake of brevity.

1. Absorbance levels were recorded every 10 min beginning from the 0^th to 60^th min in the desired wavelengths from all six blood samples. The average of the absorbances of the corresponding time intervals was plotted on a graph [Figure 1].

2. [Figure 2] illustrates the mean absorbance at different wavelengths of SK, HA, T1 and T2 at the 60^th min. [Figure 3] compares the mean peak absorbance of all samples at 405, 416, 541, 576 and 580 nm at 60^th min. Peak absorbance of SK was at 416 nm, T1 at 405 and 416 nm equally but less than HA and T2 at 405 nm. The absorbance of Sabina concentrations was more when compared to negative control HA in the higher wavelengths 541, 576 and 580 nm. However, the differences were not statistically significant.

3. The change in absorbance was highest in the 10^th min in all the samples at all wavelengths. However, the highest change in absorbance could even occur after the 10^th min and before the 20^th min, which was not recorded in this study.

4. The mean and standard deviation from the data were calculated to find the amount of dispersion for all the samples at different wavelengths. Moreover, the significance between the groups and within the groups was determined using analysis of variance [Table 1].

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Figure 1

: Comparison of mean absorbance between samples in the 1^st h at specific wavelengths.

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Figure 2:

Comparison of mean absorbance between different wavelengths of each sample.

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Figure 3:

Comparison of mean peak absorbance between samples in the 60^th min at specific wavelengths.

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DISCUSSION

In our study, we were able to partially replicate the method of assessing thrombolysis with SK adopting the protocol devised by Wang et al.^[8] The variation we observed was on the absorbance levels that rose only for the first 1 h and then gradually reached a plateau. In their experiment, the action of SK was pronounced even after 1 h, till the 3^rd h. This variation could be due to the dissimilitude between red blood corpuscles of rat and human in the metabolic pathways, cytoskeletal features of red blood cell (RBC) membrane and its transport functions.^[10] Therefore, behaviour of RBCs between different species varies and thrombolytic process would not be alike. Moreover, the SK used in this experiment is the one that is therapeutically administered for clot lysis, which could have possibly caused the radical change in thrombolysis.

Absorption peaks of Hb are observed between 200 and 900 nm of the UV-VIS spectrum due to the C = O functional group of the Hb molecule that absorbs light within the aforementioned range. This was confirmed using electron transition analysis.^[11] Due to a project constraint caused by the lack of an advanced spectrophotometer in our experiment, the release of Hb was studied only in the following wavelengths of the visible spectrum (405, 416, 541, 576 and 580 nm), which were selected after doing a literature search on the spectral activity of Hb.^[2,8,12]

The mother tincture of Sabina is prepared by mixing the coarse powder with purified water and strong alcohol.^[9,13] Therefore, the solvents are different for SK reconstitution (saline) and Sabina Q preparation (HA). As the physical and chemical properties (polarity, pH, highest occupied molecular orbital and lowest unoccupied molecular orbital transitions) vary, the optical density must be studied in the UV-VIS spectrum (190–900 nm) in all the wavelengths and especially at the isosbestic point (808–810 nm) since it will give the concentration of Hb irrespective of oxygen saturation. Due to this, a hypsochromic, hypochromic, bathochromic or hyperchromic shift can be understood if it occurs during the thrombolytic process.

Sabina Q is a heterogeneous mixture chemically composed of flavonoids, alkaloids, essential oils and many other phytoconstituents.^[13,14] To the best of our knowledge, no spectrophotometric studies or other researches are currently available showing the exact concentration of the chemical composition of Sabina Q furnishing the optical properties related to thrombolysis. In our study, the tincture was diluted to meet the recording standards of a UV/VIS spectrophotometer, as the Sabina Q solution is coloured. To circumvent this colour issue, decimal potencies may be substituted for tinctures as the colour is lighter. However, the decimal potencies require some degree of serial dilution, which will reduce the amount of phytoconstituents further to an imperceptible quantity for the interaction of Sabina Q with the clot. Hence, tincture was used in this study and the authors felt the need to devise instruments/or methods suitable to compare the strength of SK with that of concentrated Sabina Q and analyse the clot lytic activity while conducting a laboratory-based study.

Homoeopathically, Sabina is administered for menstrual haemorrhages due to its special affinity for the uterus. The haemorrhages are characteristically bright red and the flow is fluid and partly clotted. It is indicated for lingering haemorrhages or bleedings that start anew with the slightest provocation. Discharge of blood from the anus after hard evacuation has been reported. Haemorrhoids bleed with excessive bright red blood; moreover, bleeding occurs from the nose and inner parts in general. In the lower respiratory tract, haemoptysis is seen.^[3,4,15-17] Hering has cited various cases about the nature of haemorrhages where they contain a large number of clots.^[17] Since clotting is a feature associated with haemorrhage in this remedy, Sabina was selected to explore the thrombolytic property. It can be argued that bright red blood is attributed to arterial origin and thrombolysis of venous clots was studied in our research. Primarily, arterial blood is drawn to analyse the gases in critically ill patients, for placing arterial lines and in patients who undergo cardiopulmonary surgeries. As there are potential complications related to arterial blood sampling^[18] and our research was exploratory, we used venous clots. Second, menstrual blood comprises arterial and venous blood approximately in the ratio 3:1 and most importantly, fibrinolysis plays a key role to keep the menstrual blood fluid by lysing the clots that are formed.^[19] From the results of the fibrinolytic assay derived from uterine arterial and venous blood clots, fibrin degradation products were observed in higher concentrations in uterine venous blood clots than in arterial.^[20] Therefore, the results of the clot lytic activity of Sabina obtained from this study may be utilised regardless of whether the blood source is arterial or venous.

It should be stated that no conclusions can be drawn from this research regarding the use of Sabina at the bedside, as the scope of this preclinical study does not directly aid homoeopathic practice. The objective was only to evaluate the thrombolytic property. The rationale for the above conclusion is a great deal of difference between in vitro and in vivo research, especially in homoeopathy.

Homoeopathic medicines are selected according to the Similia principle and various factors such as the mental state, disposition, miasm, susceptibility, sensitivity, pace of the disease, and nature of the disease are essential to administer the remedy in the right potency to bring in a desired response. This is a foreseen disadvantage as a homoeopathic intervention on patients cannot be replicated very effectively in laboratory in vitro experiments. Nevertheless, in vitro, experiments pose an adjuvant to feed mainstream research while designing a protocol that involves human trials.^[21] Our research falls under the category of fundamental research that has provided the intended information on the thrombolytic property of Sabina that would supplement the scientific database of homoeopathic literature, which in turn may serve as a precursor for future in vivo research.

CONCLUSION

This study has shown that Sabina in the dilutions of 1:10 and 1:20 does not induce thrombolysis in vitro. By and large, further studies with different concentrations of Sabina Q with a sophisticated spectrophotometer need to be tested for thrombolytic activity.