Antitussive Effect of a Fixed Combination of Justicia Adhatoda

Phytomedicine 22 (2015) 1195–1200

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Phytomedicine

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Antitussive effect of a ﬁxed combination of Justicia adhatoda, Echinacea purpurea and Eleutherococcus senticosus extracts in patients with acute upper respiratory tract infection: A comparative, randomized, double-blind, placebo-controlled study

Anders Barth b, Areg Hovhannisyan c,∗, Kristina Jamalyan a, Mikael Narimanyan a a Yerevan State Medical University of Armenia, Koryun 2 0025, Yerevan, Armenia b Partus Kvinnohälsa, Södra v. 2, 412 54, Göteborg c Anti-doping Service of Republican Centre of Sport Medicine, Acharyan Street, 2/6, Yerevan, Armenia article info abstract

Article history: Background: Kan Jang® oral solution (KJ) is a fixed combination of aqueous ethanolic extracts of Justicia Received 20 August 2015 adhatoda L. leaf,Echinaceapurpurea(L.) Moench root, and Eleutherococcus senticosus (Rupr. & Maxim.) Harms Revised 2 October 2015 root. It is approved in Scandinavia as an herbal medicinal product for respiratory tract infection treatment. Accepted 4 October 2015 Purpose: The present clinical trial aimed to compare the antitussive effect of KJ with placebo (PL) and bromhexine (BH) among patients of 18–65 years old with non-complicated upper respiratory infections (URI; Keywords: i.e., common cold). Cough Study design: We performed a parallel-group, randomized, double-blinded, placebo-controlled trial in in 177 Kan Jang patients with acute URI over a 5 day period. Randomized controlled trial Methods: We investigated the antitussive effects of a KJ (30 ml/day; 762 mg genuine extracts with standardized contents of 0.2 mg/ml vasicine, 0.8 mg/ml chicoric acid, and 0.03 mg/ml eleutherosides B and E), bromhexine hydrochloride (24 mg/30 ml/day) and PL on cough and blood markers. The primary outcome was cough relief, which was assessed as the change of cough frequency from baseline (cough index). Secondary outcomes were safety with regards to reported adverse events (AEs) and hematological data. Results: Both KJ and BH relieved cough more effectively than placebo. On the third and fourth days of treatment, we observed faster improvement in the group receiving KJ compared to in the groups receiving BH (100%) or PL (100%), indicating a slightly shorter recovery time in the KJ group. KJ showed a good tolerability and safety profile. Conclusion: KJ exerted significant antitussive effects in URI. The present data further support the therapeutic use of KJ in upper respiratory tract infections. © 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Introduction sneeze and cough reflexes (Gwaltney and Ruckert, 1997). URIs are usually self-limiting, and thus treatments focus on reducing symp- An acute upper respiratory infection (URI) can affect the nose, si- tom duration and intensity, maximizing patient comfort, and limiting nuses, pharynx, larynx, and/or bronchi. Common URI symptoms in- complications. Cough due to URI is a frequent and disruptive symp- clude cough associated with serous or mucoid sputum (tracheitis, tom. Only limited evidence supports the few therapeutic agents avail- bronchitis); runny nose, sniffling, and nasal congestion (rhinitis); able to treat acute URI-related cough (Paul, 2012). Common cough hoarseness (laryngitis); difficulty swallowing and a sore, scratchy, treatments include narcotic antitussives, such as codeine and dex- and phlegmy throat (pharyngitis and tonsillitis); sore muscles and tromethorphan. Cough suppressants act on the brain to depress the general malaise; headache; and tiredness (Gwaltney, 2000). When cough reflex center (Reisine and Pasternak, 1996), and might be use- nasal cells are infected by a cold virus, inflammatory mediators are ful in some instances, but their efficacy is not fully proven when ex- released, stimulating nociceptors (pain nerve fibers) and activating pectoration is required (Heinrich et al., 2004). Preclinical studies show that J. adhatoda extracts have antitus- Abbreviations: KJ, Kan Jang ® oral solution; PL, placebo; BH, bromhexine. sive (Sarkar et al., 2014; Nosalova et al., 2013; Dhuley, 1999), ex- ∗ Corresponding author. Tel.: +37494 28 20 18. pectorant (Gibbs, 2009; Sharafkhaneh et al., 2007; Soni et al., 2008; E-mail address: [email protected] (A. Hovhannisyan). http://dx.doi.org/10.1016/j.phymed.2015.10.001 0944-7113/© 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1196 A. Barth et al. / Phytomedicine 22 (2015) 1195–1200

Grange and Snell, 1996), mucolytic (Gibbs, 2009; Sharafkhaneh et al., KJ compared to that of bromhexine (BH; positive control) and placebo 2007; Grange and Snell, 1996), antibacterial (Bose and Chatterjee, (PL; negative control). The study enrolled patients from the Yere- 2015; Zulqarnain et al., 2015; Singh and Sharma, 2013; Jha et al., van State Medical Centre of Armenia who presented acute uncompli- 2012; Shahwar et al., 2012; Jayashankar et al., 2011; Ignacimuthu cated URIs involving cough (ICD-10 codes J00–J06) between Novem- and Shanmugam, 2010; Gupta et al., 2010), and anti-inflammatory ber 2012 and August 2014. Inclusion criteria were age of 18–65 years (Singh and Sharma, 2013; Nosalova et al., 2013; Chakraborty and and predominant complaint of the first signs and symptoms of un- Brantner, 2001; Sharafkhaneh et al., 2007) effects. Several studies complicated URI (e.g., sore throat, blocked nose, runny nose, hoarse- describe the antitussive and respiratory effects of alkaloid vasicine ness, cough, headache, and general malaise). Patients were excluded and its oxidation product vasicinone isolated from Justicia adhatoda if they had a known history of allergies to cut flowers, herbs, or bit- L. leaves (Amin and Mehta, 1959; Cambridge et al., 1962; Mehta ter substances; had acute symptoms for over 36 hours or fever of et al., 1963; Bhide et al., 1974, 1976; Liu et al., 2015). Both in vitro and ˃38.5 °C; were taking antibiotics, anti-inflammatory drugs, or anti- in vivo studies show bronchodilatatory activity of vasicine at doses of histamine; or were pregnant or breast-feeding. Of the 180 patients 2.5–10 mg/kg, which increases with the combination of vasicine and recruited, 177 were included in the study. vasicinone. Vasicine reportedly shows the greatest activity against All included patients gave their written informed consent. The histamine-induced bronchospasm (Cambridge et al., 1962), increases study protocols were approved by the Health Research Ethics Board ciliary movements, and inhibits bronchial mucus secretion by 40–50% at the Yerevan Medical State University of Armenia (Protocol no. 01- (Gupta et al., 1977). Thus, the cough relief effects of Justicia adhatoda 2011-10-13). All patients monitoring was in compliance with the re- L. extract are mainly associated with the bronchodilation and mu- vised declaration of Helsinki. colytic effects of vasicine. However, the extract’s antitussive activity (Dhuley, 1999) is due to some other active constituents (Gupta et al., Subject withdrawals 1977). In animal studies, Justicia adhatoda L. extract shows 1/20–1/40 the antitussive activity of codeine with intravenous administration in Patients were free to withdraw from the study at any time. More- mechanically and electrically induced coughing, and antitussive ac- over, subjects were asked to withdraw from the study if they de- tivity similar to codeine with oral administration in coughing induced veloped an allergy or hypersensitivity to the study medication, de- by irritant aerosols (Dhuley, 1999). veloped serious side-effects, showed abnormal clinical biochemistry In vitro and in vivo experiments also show that Echinacea pur- values, or did not comply with the study protocol. No patients with- purea (L.) Moench, and Eleutherococcus senticosus (Rupr. & Maxim.) drew from the study. extracts activate immune system-stimulating cell defense mecha- nisms against viral and microbial infections (Echinaceae Purpureae Radix, 2009; Eleutherococci Radix, 2009). Moreover, E. senticosus in- Intervention and control treatments duces nonspecific resistance through adaptogenic effects that can ac- celerate recovery processes during convalescence (Radix Eleuthero- The KJ solution contained 9 mg/ml genuine extract (in 55% cocci, 2002). ethanol) from 23–63 mg dried root of Echinacea purpurea (L.) Moench The concepts of multi-target therapy and synergy suggest these radix (purple coneflower), 14 mg/ml genuine extract (in water) from three herbal drugs could be combined to produce a more effective 49–70 mg dried leaves of Justicia adhatoda L. folium (Malabar nut), and safe treatment, as has been demonstrated with numerous medi- and 2 mg/ml genuine extract (in 70% ethanol) from 34–60 mg cal treatments (Williamson, 2001; Wagner, 2006; Wagner and Ulrich- dried root of Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. Merzenich, 2009; Efferth and Koch, 2011). However, synergistic in- radix (Siberian ginseng). The solution base comprised sorbitol, hus- teractions of multiple constituents can be accompanied by many tenkräuter aroma, ginger extract, peppermint oil, dark syrup, ben- antagonistic and undesirable interactions (Panossian et al., 2013). zoate, and water. The preparation was standardized for contents of Therefore, clinical studies are needed to confirm the efficacy and vasicine (0.2 mg/ml), chicoric acid (0.8 mg/ml), and eleutherosides B safety of new combinations. and E (0.03 mg/ml). Validated HPLC methods were used to evaluate < Kan Jang® oral solution (KJ) is a fixed combination of aqueous selectivity, accuracy, and precision (RSD 5%) (Supplementary Fig. ethanolic extracts of Justicia adhatoda L. leaf, Echinacea purpurea (L.) 1). KJ was manufactured following GMP and released by the Swedish Moench root, and Eleutherococcus senticosus (Rupr. & Maxim.) Harms Herbal Institute Research & Development, Vallberga, Sweden (Batch root, which is approved in Scandinavia as an herbal medicinal prod- no. 2733). uct for URI treatment. A clinical study demonstrated the efficacy of The active control was the standard over-the-counter medica- KJ (30 ml taken 3 times daily for 6 days) for relief of inflammatory tion bromhexine hydrochloride (Batch no. 116075; Boehringer Ingel- symptoms (e.g., throat irritability, cough severity, efficacy of mucus heim) at a concentration of 1.6 mg/ml. This BH was dissolved in a discharge in the respiratory tract, nasal congestion, and general feel- liquid matrix containing the same excipients as the Kan Jang oral ings of sickness) among patients with acute URIs (Narimanian et al., solution: sorbitol, hustenkräuter aroma, ginger extract, peppermint 2005). That study included three arms — KJ, the combination of Echi- oil, dark syrup, benzoate, and water. The final BH concentration was nacea purpurea and Eleutherococcus senticosus, and the positive active 0.8 mg/ml (Batch no. 50136). The placebo was a mixture of sorbitol, control bromhexine hydrochloride — without a placebo group. Thus, hustenkräuter aroma, ginger extract, peppermint oil, dark syrup, ben- there remains a need to test the antitussive potential and safety of KJ zoate, and water (Batch no. 50135). Reference samples were stored at compared with placebo. the QC laboratory of the Swedish Herbal Institute. The present study aimed to evaluate the efficacy and tolerability of KJ compared to placebo and the active control bromhexine, with Allocation and blinding regard to its antitussive effect in patients with non-complicated URIs. Participants each received a labeled paper box containing either Materials and methods KJ, bromhexine, or placebo in a dark glass 300 ml bottle with a cap, sealing ring, and a measuring dosage cup (graduated at 5, 10, 15, 20, Study design and 30 ml). KJ, BH, and PL preparations all had a similar appearance, test, smell, color, test, and viscosity and were organoleptically undis- We designed a randomized, placebo-controlled, three-arm tinguishable. The trial subjects were blinded to their treatment since parallel-group, double-blind phase II trial to investigate the efficacy of the labeled bottles contained solutions of identical appearance. A. Barth et al. / Phytomedicine 22 (2015) 1195–1200 1197

Table 1 Demographic characteristics and baseline of frequency of cough of patients entered the study, n = 177.

Mean ± SD P value

KJ PL BH

Number of patients 66 54 57 KJ vs. PL KJ vs. BH Males/Females 30/36 25/29 21/36 >0.05 >0.05 Age 34.8 ± 11.39 31.9 ± 11.57 32.1 ± 10.38 >0.05 >0.05 Cough frequency 5.30 ± 0.07 5.04 ± 0.09 5.12 ± 0.08 >0.05 >0.05

To ensure that the study was double-blind, the study investigators 30 min) was expressed using a fixed scale from 0 to 9, where 0 indi- were also blinded to the treatment type using the following proce- cated no cough, 1–3 a mild cough, 4–6 a moderate cough, and 7–9 a dures. Prior to the study, a treatment randomization code was gener- severe cough. The mean cough frequency value at baseline was 5.0– ated at the factory, which contained information about the contents 5.3, indicating a moderate cough (Table 1). Patients were instructed of each bottle, e.g., encoding KJ, BH, or PL. This code was generated to immediately contact the investigator should they experience any using the Excel Random Number Generator, with a table containing study events, defined as any relevant event experienced by the pa- three columns (A, B, and C) that was filled with randomly distributed tient during the study. Given examples included adverse effects and unique numbers from 1 to 198. KJ, PL, and BH were assigned to sets of inter-current illness. In the event of any allergic reactions, such as bottles A, B, and C at the manufacturing site. The treatment random- skin rash, the medication would be stopped. ization code was kept by a qualified pharmacist (QP) at the investiga- In addition to study events, safety was monitored using hemato- tional product manufacturing site, and was provided to the Principal logical data. Laboratory blood analyses were performed using sam- Investigator when all patients completed the treatment. ples from day 1 and day 5. Analyses included hemoglobin, red blood Participants were enrolled by the principal investigator in collab- cell count (RBC), color index, neutrophil count, basophil count, eosin oration with study doctors. The study monitor assigned patients to plates, and white blood cells (WBC), and were performed using an study groups A, B, and C. The study monitor did not receive the ran- Automated Hematology Analyzer pocH-100i (Sysmex Corporation, domization code until the end of the study. The Principal Investiga- Cobe, Japan). tor generated the Study Participants list, identifying the patients and the study supplement packages (treatment numbers). At the end of Sample size considerations the study, statistical analyses were performed using the Study Partic- ipants list to compare the data obtained in groups A, B, and C. Fol- The sample size for the present study was calculated based on an lowing these statistical analyses, the QP broke the code providing the effect size of 1.42 from a comparable study of KJ in URIs, in which actual assignments of groups A, B, and C. cough and other inflammatory symptoms were used as efficacy outcome measures (Narimanian et al., 2005). Using a two sided, two- sample t-test, it was calculated that 30 participants in each group Patient intervention and examinations were required to detect an absolute mean difference of 1.42 on the cough relief score between the KJ and BH groups with 95% power at At visit 1 (Supplementary Fig. 2), patients underwent a routine a 0.05 level of significance (Stat-Mate, version 2.00, 2004; GraphPad medical examination that included physical examinations (anamne- Software, Inc.). We prepared for a 20% drop-out rate, increasing our sis collection, detailed chest auscultation and percussion, and ther- intended sample size to 118 participants (36 in each group). In the mometry) and laboratory blood hematological tests. During this ini- completed trial, we had over 99% power to detect a between-means tial examination, the doctor counted the cough frequency (number difference of 0.28 with a two-tailed alpha of 0.05: KJ, n = 66, SD = of coughing bouts within 30 min), recorded the results in the Case 0.43; BH, n = 57, SD = 0.40; and PL, n = 54, SD = 0.28. Report Form (CRF), and instructed patents in how self-assess their cough frequency at home. Each participant was provided a report Statistical analysis form, and was instructed to count the number of coughing bouts for 30 min following the first coughing incident in the morning immedi- At each visit, data were recorded using a standardized assess- ately after waking up. ment method, and recorded in an Excel database that was used for Patients who passed their initial examination and gave informed further data management and statistical analyses (Evererett, 1989). consent to participate were then assigned to a randomized study GraphPad Prism software version 3.03 for Windows (San Diego, CA, medication. They were provided this medication and asked to take USA) was used for statistical analyses and graph generation. The pri- 30 ml of oral solution per day for the next 5 days. The medication mary analysis followed intention-to-treat principles. Statistical anal- was to be taken 30 min after eating breakfast. The patients were also yses were performed using “observed” data, and involved evaluating instructed to regularly record the degree of their clinical symptoms the patient’s change in scores from the initial visit (baseline) to later and the amount of medication taken daily on a patient’s-self assess- days of the study and the final visit. All statistical tests were two- ment forms. At the end of the study (visit 2, day 5), the patients again sided, with a 0.05 level of significance. met with a doctor. At this visit, the doctor checked compliance (liquid Before within-group or between-group comparisons, all data consumption) and checked and signed the patient’s completed forms. were checked for normality, with a cut-off value of 0.05. Based on the normality test results, comparative assessment of the baseline Outcomes characteristics between groups (KJ vs. PL and BH) was performed using either the Kruskal–Wallis non-parametric one-way ANOVA rank- The primary outcome was efficacy of treatment (Cough Relief In- order test, with post-hoc Dunn’s Multiple Comparison Test or the dex), which was determined based on the difference between the parametric one-way independent measures ANOVA with Dunnett’s cough frequency on day 1 (baseline, determined by the physician at Multiple Comparison Test. Changes within treatment groups over the visit 1) and on each successive day of treatment (determined by the course of the study (before vs. after) were analyzed using the paired patient at home). Cough frequency (the number of coughing bouts in t-test for parametric data (variables with normal distribution) and 1198 A. Barth et al. / Phytomedicine 22 (2015) 1195–1200

Table 2 Safety Baseline cough frequency scores at the beginning (day 1) and the end (day 5) of the study. The treatment was well tolerated. No serious adverse events were Baseline score Day 5 score observed. Over the 5 day study period, a total of 12 minor adverse Score KJ PL BH Score KJ PL BH events were observed in 12 patients: 4 in the PL group (7.4%), 2 in the KJ group (3.9%), and 6 in the BH group (10.5%). Minor adverse 73004240 events included pruritus, diarrhea, abdominal pain, and skin rash. The 62620263394 5 70 65 60 2 29 31 40 proportion of patients reporting adverse events did not signiﬁcantly 4213141654844 differ between groups, and these events were not considered to be 302002712 treatment-related.

Hematological and immunological parameters the Wilcoxon signed-rank test or the Friedman test for several re- peated measures for non-parametric data. To assess the efficacy of the The only statistically significant between-group difference at the study supplements, we compared the mean changes from baseline end of the study (day 5) was that lymphocyte count differed between for each patient and each group (KJ vs. PL and BH) using the Kruskal– the BH and KJ groups (Table 5). Wallis non-parametric one-way ANOVA rank-order test, with post- hoc Dunn’s Multiple Comparison Test, and/or the parametric one-way independent measures ANOVA with Dunnett’s Multiple Comparison Discussion Test (for variables with normal distribution). Kan Jang® is a brand of herbal medicinal products that are used in Scandinavia to treat URIs. Previous trials of drugs in various stages of Results development have investigated formulations containing Herba Adro- graphidis extract, including a monodrug (Hancke et al., 1995; Cac- Study participants and baseline variables eres et al., 1997) and fixed combinations of Herba Andrographidis extract with Radix Eleutherococci (Melchior et al., 2000; Gabrielian A total of 180 subjects were recruited and 177 patients were in- et al., 2002; Kulichenko et al., 2003; Poolsup et al., 2004; Kligler cluded in this study. These patients were randomly divided into three et al., 2006) or Radix Echinacea extracts (Spasov et al.,2004). Herbal groups. Participants flow is outlined in Supplementary Fig. 3. Table 1 tablets or capsules with standardized andrographolide content are presents the baseline demographic and clinical characteristics of each mainly indicated for early prevention and relief of the symptoms of group and the relevant baseline data. The groups did not significantly URIs. differ in baseline demographics or other parameters (Table 1). Another group of Kan Jang formulations includes fixed combinations of Folia Adhatoda extracts with extracts of Radix Eleutherococci Treatment efficacy and E. pallida (Thom and Wollan, 1997)orE. purpurea (Narimanian et al., 2005). Oral solutions of these fixed combinations are indicated Over the course of the study, each group showed significant relief for cough relief, with the efficacy presumably due to the antitus- of cough compared to the baseline and/or each previous day (Tables 2 sive, bronchodilating, and expectorant effects of the alkaloid vasicine and 3). (Amin and Mehta, 1959; Cambridge et al., 1962; Mehta et al., 1963; Cough relief was measured as the change of the cough index from Bhide et al., 1974, 1976; Liu et al., 2015), which is an active constituent baseline. On the third and fourth days of treatment, we found a signif- of J. adhatoda leaf extract. Its semi-synthetic derivative bromhexine icantly greater cough relief effect with KJ treatment compared to PL (Bisolvon) (Grange and Snell, 1996)iswidelyusedasanover-the- and BH (Table 4, Supplementary Fig. 4). On the third treatment day, counter mucolytic medication to reduce cough (Parvez et al., 1996), the cough relief effect was 1.97 ± 0.021 in the KJ group, compared particularly as an adjunct to antibiotics for acute pneumonia in chil- to 1.759 ± 0.079 in the placebo group (P < 0.05) and 1.772 ± 0.083 dren and adults (Chang et al., 2014). in the BH group (P < 0.05). The difference in effect and the statisti- The present clinical trial investigated the antitussive effect of a cal significance of the difference between the KJ and BH groups was Kan Jang® oral solution containing a fixed combination of aqueous maximal on day 4 (P < 0.01). At the end of treatment (day 5), all pa- and ethanolic extracts of Justicia adhatoda L. leaf, Echinacea purpurea tients had recovered, and the cough relief effects were similar in the (L.) Moench root, and Eleutherococcus senticosus (Rupr. & Maxim.) KJ and BH groups (P > 0.05). Our data also showed a greater speed Harms root extracts (KJ). Our randomized double-blind three-arm of recovery (assessed as cough relief effect) for patients in the KJ and study aimed to compare the efficacy and tolerability of KJ with a positive control groups compared to in the placebo group. placebo and with the active control BH with respect to the antitussive

Table 3 Cough frequency in the course of the study and cough relief (Cough index) at the end of the treatment: within group comparison.

KJ PL BH n = 66 n = 54 n = 57 mean ± SD mean ± SD mean ± SD

Baseline, Day 1 5.303 ± 0.554 5.037 ± 0.643 5.140 ± 0.666 Day 2 4.333 ± 0.591 4.111 ± 0.691 4.281 ± 0.726 Day 3 3.333 ± 0.591 3.278 ± 0.787 3.368 ± 0.858 Day 4 2.364 ± 0.598 2.463 ± 0.884 2.526 ± 0.966 Day 5 1.379 ± 0.651 1.537 ± 0.905 1.351 ± 0.744 Cough index on the day 5: change from baseline, P value 3.924 ± 0.1052 3.500 ± 0.1511 3.772 ± 0.1290 <0.0001 <0.0001 <0.0001

∗The intensity of cough measured as a cough index between groups did not show any signiﬁcant difference at the start. A. Barth et al. / Phytomedicine 22 (2015) 1195–1200 1199

Table 4 Eﬃcacy of treatment of the frequency of cough: cough index (cough relief effect form the baseline, day 1) in treatment groups in the course of the treatment: between groups comparison.

KJ PL BH KJ vs. PL KJ vs. BH n = 66 n = 54 n = 57 P value and summary P value and summary mean ± SD mean ± SD mean ± SD

Day 2 0.970 ± 0.173 0.926 ± 0.381 0.860 ± 0.398 >0.05ns >0.05ns Day 3 1.97 ± 0.172 1.759 ± 0.581 1.772 ± 0.627 <0.05∗ <0.05∗ Day 4 2.939 ± 0.241 2.574 ± 0.716 2.614 ± 0.773 <0.01∗∗ <0.01∗∗ Day 5 3.924 ± 0.404 3.500 ± 0.795 3.772 ± 0.567 <0.001∗∗∗ >0.05ns

Table 5 Inter-groups comparison of hematological/immunological parameters.

KJ PL BH KJ vs. PL KJ vs. BH n = 66 n = 54 n = 57 P value P value mean ± SD mean ± SD mean ± SD Hemoglobin, (g/l) [Norm – 120–160 g/l]

Baseline 127.1 ± 13.31 128.2 ± 13.06 124.3 ± 14.61 >0.05 >0.05 Day 5 126.4 ± 13.23 127.6 ± 12.88 123.7 ± 14.34 >0.05 >0.05 Change from baseline 0.677 ± 2.310 0.531 ± 2.496 0.568 ± 2.711 >0.05 >0.05 RBC, (10∗12/l) [Norm – 3.5–5.0 1012 /l] Baseline 3.718 ± 0.387 3.752 ± 0.367 3.626 ± 0.423 >0.05 >0.05 Day 5 3.644 ± 0.366 3.687 ± 0.338 3.567 ± 0.408 >0.05 >0.05 Change from baseline 0.074 ± 0.065 0.065 ± 0.068 0.060 ± 0.078 >0.05 >0.05 Color Index, [Norm – 0.85–1.05]

Baseline 0.942 ± 0.049 0.943 ± 0.05 0.939 ± 0.049 >0.05 >0.05 Day 5 0.945 ± 0.050 0.946 ± 0.05 0.940 ± 0.049 >0.05 >0.05 Change from baseline −0.003 ± 0.009 −0.004 ± 0.01 −0.002 ± 0.009 >0.05 >0.05 Neutrophils, % [Norm – 1–6%]

Baseline 3.045 ± 1.513 3.037 ± 1.479 3.228 ± 1.476 >0.05 >0.05 Day 5 2.530 ± 1.14 2.519 ± 1.285 2.649 ± 1.275 >0.05 >0.05 Change from baseline 0.561 ± 0.096 0.518 ± 0.267 0.579 ± 0.258 >0.05 >0.05 Basophils, % [Norm – 47–72%]

Baseline 64.08 ± 4.368 64.09 ± 4.127 63.79 ± 3.261 >0.05 >0.05 Day 5 62.97 ± 4.325 62.70 ± 3.451 62.74 ± 2.955 >0.05 >0.05 Change from baseline 1.106 ± 0.785 1.389 ± 0.732 1.053 ± 0.583 >0.05 >0.05 Eosinophils, % [Norm – 0.5–5.0%]

Baseline. 3.515 ± 0.662 3.426 ± 0.716 3.614 ± 0.675 >0.05 >0.05 Day 5 2.955 ± 0.409 2.870 ± 0.516 2.930 ± 0.495 >0.05 >0.05 Change from baseline 0.5606 ± 0.096 0.556 ± 0.120 0.684 ± 0.110 >0.05 >0.05 Lymphocytes, %, [Norm –19–37%]

Baseline 24.24 ± 4.581 25.22 ± 4.756 24.21 ± 5.460 >0.05 >0.05 Day 5 23.38 ± 4.205 23.37 ± 5.275 23.21 ± 5.02 >0.05 >0.05 Change from baseline 0.864 ± 0.765 1.852 ± 0.967 1.000 ± 0.982 >0.05 >0.05 Monocytes, % [Norm – 2.9–11 %]

Baseline 3.045 ± 0.325 2.963 ± 0.433 2.947 ± 0.440 >0.05 >0.05 Day 5 3.000 ± 0.248 2.907 ± 0.293 2.930 ± 0.258 >0.05 >0.05 Change from baseline 0.045 ± 0.050 0.055 ± 0.071 0.017 ± 0.067 >0.05 >0.05 Erythrocytes sedimentation rate (ESR). mm/h [Norm – 2–20 mm/h]

Baseline 20.74 ± 4.531 19.26 ± 4.639 19.98 ± 4.725 >0.05 >0.05 Day 5 19.21 ± 4.219 17.33 ± 4.421 16.12 ± 4.886 >0.05 >0.05 Change from baseline 1.530 ± 0.762 1.926 ± 0.872 2.140 ± 0.9 >0.05 >0.05 Whyte blood cells, WBC (10∗9/L) [Norm –4–9.0 109/l]

Baseline 8.524 ± 0.581 8.287 ± 0.673 8.287 ± 0.759 >0.05 >0.05 Day 5 8.232 ± 0.612 8.023 ± 0.640 8.023 ± 0.696 >0.05 < 0.05∗ Change from baseline 0.292 ± 0.104 0.264 ± 0.127 0.264 ± 0.128 >0.05 >0.05 effect. The efficacy was analyzed based on patient self-evaluations us- group showed a greater reduction of cough intensity compared ing a standard rating scale from 0–9 to indicate cough frequency and to both the PL and BH groups, indicating a quicker recovery in intensity. the KJ group. All treatments were well tolerated, with no seri- The three treatment groups did not significantly differ at base- ous adverse events, and only 12 minor adverse events, including line or at the end of treatment when all patients were almost mild pruritus in 6 patients (2 in each group). Overall, our data fully recovered. On the second day of treatment, we observed showed that KJ exerted significantly more rapid antitussive effects significant improvement and antitussive effect in all treatment in acute upper respiratory tract infections compared to both BH and groups (including placebo), which gradually increased over the fol- placebo. Moreover, KJ showed good tolerability and a good safety lowing days. On the third and fourth days of treatment, the KJ profile. 1200 A. Barth et al. / Phytomedicine 22 (2015) 1195–1200

Conclusion Heinrich, M., Barnes, J., Gibbons, S., Williamson, E.M., 2004. Fundamentals of Phar- macognosy & Phytotherapy. Churchil Livingstone, Edinburg-London-NewYork- Oxford-Philadelphia-StLuis-Sydney-Toronto. Kan Jang® oral solution exhibits significant antitussive effects Ignacimuthu, S., Shanmugam, N., 2010. Antimycobacterial activity of two natural alka- in acute upper respiratory tract infections. It has good tolerability loids, vasicine acetate and 2-acetyl benzylamine, isolated from Indian shrub Adha- profile. toda vasica Ness. leaves. J Biosci. 35, 565–570. Jayashankar, S., Panagoda, G.J., Amaratunga, E.A., Perera, K., Rajapakse, P.S., 2011. A randomized double-blind placebo-controlled study on the effects of a herbal tooth- Conflict of interest paste on gingival bleeding, oral hygiene and microbial variables. Ceylon Med J. 56, 5–9. Jha, D.K., Panda, L., Lavanya, P., Ramaiah, S., Anbarasu, A., 2012. Detection and confir- No conflict of interests declared. mation of alkaloids in leaves of Justicia adhatoda and bioinformatics approach to elicit its anti-tuberculosis activity. Appl Biochem Biotechnol. 168, 980–990. Kligler, B., Ulbricht, C., Basch, E., Kirkwood, C.D., Abrams, T.R., Miranda, M., Singh Acknowledgments Khalsa, K.P., Giles, M., Boon, H., Woods, J., 2006. Andrographis paniculata for the treatment of upper respiratory infection: a systematic review by the natural stan- This work was supported in part by the Swedish Herbal Institute dard research collaboration. Explore (NY) 2 (1), 25–29. Kulichenko, L.L., Kireyeva, L.V., Malyshkina, E.N., Wikman, G., 2003. A randomized, con- Research & Development. trolled study of Kan Jang versus amantadine in the treatment of influenza in Vol- gograd. J Herb Pharmacother 3 (1), 77–93 PubMed PMID: 15277072. Supplementary materials Liu, W., Wang, Y., He, D.-D., Li S.-P., Zhu, Y.-D., Jiang, B., Cheng, X.-M., Wang Z.-T., Wang C.-H. 2015. Antitussive, expectorant, and bronchodilating effects of quina- zoline alkaloids (±)-vasicine, deoxyvasicine, and (±)-vasicinone from aerial parts Supplementary material associated with this article can be found, of Peganum harmala L Phytomedicine, Available online 28 August 2015. http: in the online version, at doi:10.1016/j.phymed.2015.10.001. //dx.doi.org/10.1016/j.phymed.2015.08.005 Mehta, D.R., Naravane, J.S., Desai, R.M., 1963. Vasicinone. A bronchodilator principle from Adhatoda vasica Nees (N. O. Acanthaceae). J. Organic Chem. 28, 445–448. References Melchior, J., Spasov, A.A., Ostrovskij, O.V., Bulanov, A.E., Wikman, G., 2000. Double- blind, placebo-controlled pilot and phase III study of activity of standardized An- Amin, A.H., Mehta, D.R., 1959. A bronchodilator alkaloid (vasicinone) from Adhatoda drographis paniculata Herba Nees extract fixed combination (Kan jang) in the vasica Nees. Nature 18, 1317. treatment of uncomplicated upper-respiratory tract infection. Phytomedicine 7 Bhide, M.B., Naik, P.Y., Ghool, R.B., 1976. Comparative studies on the pharmacological (5), 341–350. evaluation of vasicine and vasicinone. Bull. Haffkine Inst. 4, 43–50. Narimanian, M., Badalyan, M., Panosyan, V., Gabrielyan, E., Panossian, A., Wikman, G., Bhide, M.B., Naik, P.Y., Mahajani, S.S., Ghool, R.B., Joshi, R.S., 1974. Pharmacological eval- Wagner, H., 2005. Clinical evidence of high efficacy of fixed combinations of uation of vasicinone. Bull. Haffkine Inst. 2, 6–11. herbal extracts, with particular reference to Adhatoda vasica, in patients with Bose, D., Chatterjee, S., 2015. Antibacterial activity of green synthesized silver nanopar- non-complicated respiratory tract infections (bronchitis). Phytomedicine 12, 539– ticles using vasaka (Justicia adhatoda L.) leaf extract. Indian J Microbiol. 55, 163– 547. 167. Nosalova, G., Fleskova, D., Jurecek, L., Sadlonova, V., Ray, B., 2013. Herbal polysaccha- Caceres, DD, Hancke, JL, Burgos, RA, Wikman, GK, 1997 Jun. Prevention of common rides and cough reflex. Respir Phys. Neurobiol. 187, 47–51. colds with Andrographis paniculata dried extract. A Pilot double blind trial. Phy- Parvez, L., Vaidya, M., Sakhardande, A., Subburaj, S., Rajagopalan, T.G., 1996 Oct-Dec. tomedicine. 4 (2), 101–104. Evaluation of antitussive agents in man. Pulm Pharmacol. 9 (5–6), 299–308. Cambridge, G.W., Jansen, A.B.A., Jarman, D.A., 1962. Bronchodilating action of vasici- Paul, I.M., 2012. Therapeutic options for acute cough due to upper respiratory infec- none and related compounds. Nature 4860, 1217. tions in children. Lung 190, 41–44. Chakraborty, A., Brantner, A.H., 2001. Study of alkaloids from Adhatoda vasica Nees on Poolsup, N., Suthisisang, C., Prathanturarug, S., Asawamekin, A., Chanchareon, U., 2004. their antiinflammatory activity. Phytother Res. 15, 532–534. Andrographis paniculata in the symptomatic treatment of uncomplicated upper Chang, CC, Cheng, AC, Chang, AB, 2014 Mar 10. Over-the-counter (OTC) medications respiratory tract infection: systematic review of randomized controlled trials. J Clin to reduce cough as an adjunct to antibiotics for acute pneumonia in children and Pharm Ther. 29 (1), 37–45. adults. Cochrane Database Syst Rev. 3, CD006088. Radix Eleutherococci, 2002. WHO Monographs on Selected Medicinal Plants – Volume Dhuley, J.N., 1999. Antitussive effect of Adhatoda vasica extract on mechanical or chem- 2. WHO, Geneva, pp. 83–96. ical stimulation-induced coughing in animals. J Ethnopharmacol. 67, 361–365. Reisine, T., Pasternak, G., 1996. Opioid analgesics and antagonists. In: Hardman, J.G., Echinaceae Purpureae Radix. Purple Coneflower Root, 2009. ESCOP Monographs: The Limbird, L.E., Molinoff, P.B., Ruddon, R.W., Gilman, A.G. (Eds.), Goodman and Scientific Foundation for Herbal Medicinal Products, second edition. European Sci- Gilman’s the pharmacological basis of therapeutics. McGraw Hill, New York, entific Cooperative on Phytotherapy and Thieme, Exeter (GB), pp. 87–91 Supple- pp. 521–555. ment 2009. Sarkar, C., Bose, S., Banerjee, S., 2014. Evaluation of hepatoprotective activity of vasici- Efferth, T., Koch, E., 2011. Complex interactions between phytochemicals. The multi- none in mice. Indian J Exp Biol. 52, 705–711. target therapeutic concept of phytotherapy. Curr. Drug Targets 12, 122–132. Shahwar, D., Raza, M.A, Tariq, S., Riasat, M., Ajaib, M., 2012. Enzyme inhibition, antiox- Eleutherococci Radix. Eleuthrococcus, 2009. ESCOP Monographs: The scientific foun- idant and antibacterial potential of vasicine isolated from Adhatoda vasica Nees. dation for herbal medicinal products, second edition. European Scientific Cooper- Pak JPharm Sci. 25, 651–656. ative on Phytotherapy and Thieme, Exeter (GB), pp. 110–120 Supplement 2009. Sharafkhaneh, A., Velamuri, S., Badmaev, V., Lan, C., Hanania, N., 2007. The potential Evererett, B.S., 1989. Statistical Methods for Medica Investigations. Oxford University role of natural agents in treatment of airway inflammation. Ther. Adv. Respir. Dis. Press, New York. 1, 105–120. Gabrielian, E.S., Shukarian, A.K., Goukasova, G.I., et al., 2002. A double blind, placebo- Singh, B., Sharma, R.A., 2013. Anti-inflammatory and antimicrobial properties of controlled study of Andrographis paniculata fixed combination Kan Jang in the pyrroloquinazoline alkaloids from Adhatoda vasica Nees. Phytomedicine 20, 441– treatment of acute upper respiratory tract infections including sinusitis. Phy- 445. tomedicine 9 (7), 589–597. Soni, S., Anandjiwala, S., Patel, G., Rajani, M., 2008. Validation of different methods of Gibbs, B.F., 2009. Differential modulation of IgE-dependent activation of human ba- preparation of adhatoda vasica leaf juice by quantification of total alkaloids and sophils by ambroxol and related secretolytic analogues. Int J Immunopathol Phar- vasicine. Indian J Pharm Sci. 70, 36–42. macol. 22, 919–927. Spasov, A.A., Ostrovskij, O.V., Chernikov, M.V., et al., 2004. Comparative controlled Grange, J.M., Snell, N.J., 1996. Activity of bromhexine and ambroxol, semi-synthetic study of Andrographis paniculata fixed combination, Kan Jang® and an echinacea derivatives of vasicine from the Indian shrub Adhatoda vasica, against Mycobac- preparation as adjuvant, in the treatment of uncomplicated respiratory disease in terium tuberculosis in vitro. J Ethnopharmacol. 50, 49–53. children. Phytotherapy Res. 18 (1), 47–53. Gupta, O.P., Sharma, M.L., Ray Ghatak, B.J., Atal, C.K., 1977. Pharmacological investiga- Thom, E., Wollan, T., 1997. A controlled clinical study of Kanjang mixture in the treat- tions of vasicine and vasicinone – the alkaloids of Adhatoda vasica. Indian J Med ment of uncomplicated upper respiratory tract infections. Phytotherapy Res. 2, Res 66, 680–691. 207–210. Gupta, R., Thakur, B., Singh, P., Singh, H.B., Sharma, V.D., Katoch, V.M., Chauhan, S.V., Wagner, H., 2006. Multitarget therapy–the future of treatment for more than just func- 2010. Anti-tuberculosis activity of selected medicinal plants against multi-drug tional dyspepsia. Phytomedicine 13, 122–129. resistant Mycobacterium tuberculosis isolates. Indian J Med Res. 131, 809– Wagner, H., Ulrich-Merzenich, G., 2009. Synergy research: approaching a new genera- 813. tion of phytopharmaceuticals. Phytomedicine. 16, 97–110. Gwaltney Jr., J.M., 2000. The common cold. In: Mandell, G.L., Bennett, J.E., Dolin, R. Williamson, E.M., 2001. Synergy and other interactions in phytomedicines. Phy- (Eds.), Principles and practices of infectious diseases. Churchill Livingstone, New tomedicine 8, 401–409. York, pp. 651–656. Zulqarnain, R.A., Ahmad, K., Ullah, F., Ullah, H., Nishan, U., 2015. In vitro antibacterial Gwaltney Jr., J.M., Ruckert, R.R., 1997. Rhinovirus. In: Richman, D.D., Whitley, R.J., Hay- activity of selected medicinal plants from lower Himalayas. Pak J Pharm S. 28, 581– den, F.G. (Eds.), Clinical Virology. Churhill Livingstone, New York, pp. 1025–1047. 587. Hancke, J., Burgos, R., Caceres, D., et al., 1995. A double-blind study with a new monodrug Kan Jang: decrease of symptoms and improvement in the recovery from common colds. Phytotherapy Research 9, 559–562.