What is Ashwagandha?

Withania somnifera (Ashwagandha)
Analytical Method Development
Sanni Raju, Ph.D., R.Ph.
Natreon, Inc.
2D - Janine Place, New Brunswick, NJ
08901
Tel: 732-296-1080; Fax: 732-777-5129
E-mail: [email protected]
Website: www.natreoninc.com
1
What is Ashwagandha?




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
Ashwagandha is an Ayurvedic medicine derived from the plant
Withania somnifera.
A ti t
Anti-stress
Improves endothelial function
Cognition enhancing
Anti-inflammatory
Lipid management
CONFIDENTIAL
2
Fitness for Purpose Statement





Method should be able to quantitatively analyze withanolide glycosides,
W h f
Withaferin-A
A and
d oligosaccharides
l
h d from
f
nutraceuticall matrices, such
h as powders,
d
tablets, capsules and liquids.
Method is confirmatory and will be used to pass or fail a batch of the raw
material (powder extract) or a finished product, such as a tablet or a capsule.
There is no regulatory requirement
The method will be used in a laboratory set up.
Chemists trained in HPLC/HPTLC techniques will be required to do the analysis.
3
Fitness for Purpose Statement
Points for discussion by SPDS




How does this method address Ashwagandha extracts made by different
manufacturers
What should be the targeted analytes
Limits of quantification
Availability of reference standards
4
Extraction Methodology
Traditionally only roots have been used
Natreon developed a better extract using roots plus leaves
(patented)
 Most companies use either alcoholic or hydro-alcoholic
solvents for extraction.
 One company has used supercritical extraction using carbon
dioxide.
 Natreon’s method is completely aqueous.
 Depending on the extraction method used, chemical
constitution of the extract varies.


CONFIDENTIAL
5
Chemical Composition
Depending on the extraction solvent and the
method used, the Ashwagandha extract may contain any
off th
the ffollowing
ll i constituents:
tit
t
o Withanolide glycosides
o Withaferin A
o Oligosaccharides
o Alkaloids
o Polysaccharides
y
o Withanostramnolide
o 27-hydroxy withanone
o 27-hydroxy withanolide B
CONFIDENTIAL
6
Intellectual Property - Sensoril®


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

US 7,318,938: Withania somnifera composition, method for
obtaining same and pharmaceutical, nutritional and personal care
formulations thereof
US 6,713,092 B1: Withania somnifera composition, method for
obtaining same and pharmaceutical, nutritional and personal care
formulations thereof
US 6,153,198: Withania somnifera composition
EP1569669 A2:Withania somnifera composition, method for
obtaining same and pharmaceutical, nutritional and personal care
formulations thereof
Canadian Patent No. 2,508,478
,
,
– Withania somnifera composition,
p
,
method for obtaining same and pharmaceutical, nutritional and
personal care formulations thereof
US 8,206,757: Combination of Ashwagandha and Indian
Gooseberry
Other pending US and PCT applications
CONFIDENTIAL
7
Human clinical study of Sensoril® for stress
management
o Number of patients
o Randomized,
Randomized placebo
placebo-controlled
controlled, double blind study
o 130 chronically stressed subjects enrolled; 98 completed
o Treatment schedule
®
• Group 1: Sensoril (250 mg capsule; 2/day)
• Enrolled 35; Completed 34
®
• Group 2: Sensoril (125 mg capsule; 2/day)
• Enrolled 35; Completed 30
• Group 3: Sensoril® (125 mg capsule; 1/day)
• Enrolled 30; Completed 19
• Group 4: Placebo (2/day)
• Enrolled 30; Completed 15
• Comprehensive assessments done at
0 day, after 30 and 60 days treatment
Published: JANA, Vol. II, No.1, 208, Pages 50-56.
CONFIDENTIAL
8
Clinical study results – Stress & Anxiety
parameters

Hamilton Anxiety Scale

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
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Pulse rate
rate*
Blood pressure*
Sleep deprivation*
Palpitation*
Dry mouth*
Perspiration*
Appetite
Fatigue*
Flushes*
Headache & Muscular pain*
Forgetfulness*
Irritability*
Inability to concentrate*
Feeling of impending
doom*

Scoring system (subjective)
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Statistical significance

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
Severe – 4
Moderate – 3
Mild – 2
Occasional – 1
None – 0
ANOVA
P <0.05 (*significant)
Conclusion

Statistically significant
reduction seen in all the 12
stress & anxiety and 2
quantitative parameters
CONFIDENTIAL
9
Clinical study results – % Change in gross
stress & anxiety parameters
% Reduction in gross stress condition in 30 & 60-day treatment
30
30
60
30
60
60
30 60
30 day
p‐values <0.001
p
CONFIDENTIAL
10
Clinical study results: Stress markers
% Reduction in serum cortisol
% Reduction in C-reactive protein
% Improvement in serum DHEAS
Statistical significance
• Day 0 vs 60 day; ANOVA
• 250 mg & 125 mg BID: p <0.001
• 125 mg OD: p <0.01
CONFIDENTIAL
11
Clinical study results: Lipid profile
% Reduction in cholesterol
% Reduction in triglycerides
% Reduction in LDL
% Improvement in HDL
CONFIDENTIAL
12
Clinical study results: Effects on VLDL,
hemoglobin & glucose
% Reduction in VLDL
% Improvement in Hemoglobin
% Reduction in glucose
Statistical significance
• Statistical significance only with 250 mg BID
• One way ANOVA; p <0.001
Nonsignificant
CONFIDENTIAL
13
Comparison of Sensoril® data to Lexapro® and Effexor® data
from literature - HAM-A/mHAM-A Day 0 – Day 60
Hamilton parameter: Initial vs 60-day
CONFIDENTIAL
14
Sensoril® - Cardiovascular/Endothelial
Dysfunction Clinical Study

Study Design
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Inclusion Criteria
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Prospective, randomized, double blind study with placebo and positive
(Atorvastatin/Lipitor®) controls with 20 subjects in each group.
Patients with Type 2 diabetes stabilized on Metformin treatment
Patients of either sex, aged 18-75 years
Fasting plasma glucose of ≥110 mg/dL
Glycosylated haemoglobin (HbA1c) between 7 % and 9%
Taking stable dose of anti-diabetic treatment for the past 8 weeks prior to the
screening visit
Endothelial dysfunction defined as ≤ 6% change in reflection index (RI) on post
salbutamol challenge test
Exclusion Criteria

Patients with severe uncontrolled hyperglyceamia, uncontrolled hypertension,
cardiac arrhythmia, impaired hepatic or renal function, history of malignancy or
stroke, smoking, chronic alcoholism, any other serious disease requiring active
treatment and treatment with any other herbal supplements and pregnant and
lactating women.
CONFIDENTIAL
15
Sensoril® - Cardiovascular/Endothelial Dysfunction
Clinical Study – Demographics & Dosage Regimen
Parameter
Placebo
Sensoril
250 mg
Sensoril
500 mg
Atorvastatin
20
20
20
20
57.45±8.85
55.40±8.07
57.30±9.40
56.95±8.04
12/8
14/6
13/7
13/7
66.09±5.56
68.07±6.51
67.30±6.16
68.56±8.47
24.82±1.86
24.89±2.03
25.01±2.92
26.02±3.12
N
N
Age
Sex (M/F)
Weight (Kg)
BMI (Kg/m2)
Duration: 12 weeks
Sensoril®: 250mg and 500 mg twice daily
Atorvastatin: One capsule of Atorvastatin 10mg (night) and one capsule of Placebo (morning)
Placebo: One capsule of placebo twice daily
CONFIDENTIAL
16
Sensoril® treatment improves endothelial
function
Refelection Index: A salbutamol challenge test employing digital volume
plethysmography was used to assess endothelial function as reported by Chowienczyk et
al (J Am Coll Cardiol, 34(7):2007-2014, 1999) and Naidu et al (Indian J
Pharmacol,39:168-169, 2007)
CONFIDENTIAL
17
Sensoril® treatment improves endothelial
function
*
**
**
**
**
**
CONFIDENTIAL
18
Sensoril® treatment reduces cardiovascular risk
factors
*
**
**
**
**
**
CONFIDENTIAL
19
Sensoril® treatment reduces cardiovascular risk
factors
*
**
**
**
**
**
CONFIDENTIAL
20
Sensoril® treatment reduces cardiovascular risk
factors
*
**
**
**
**
**
P=<0.05
CONFIDENTIAL
21
% Change
Sensoril® treatment reduces cardiovascular risk
factors
*
**
**
**
1
**
**
3
1 p<0.01 when compared to Placebo
2 p<0.001 when compared to Placebo
3 p<0.001 when compared to Placebo
CONFIDENTIAL
22
Sensoril® - Clinical Study - Mental Cognition in Stable
Bipolar Patients – University of Pittsburgh

Study Design: As per IND with US FDA
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Dosage Regimen
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Double blind, randomized, parallel group, adjunctive treatment, 30
subjects
j
in each g
group.
p
Sensoril® vs. Placebo, added to ongoing maintenance bipolar
medications
250 mg first week, once a day, increased to 250 mg twice daily, for rest
of the study
Total Duration: 8 weeks
Funded by

NARSAD (NY based sponsor of studies in bipolar and schizophrenic
patients)
Accepted for publication in the Journal of
Clinical Psychiatry
CONFIDENTIAL
23
Sensoril® - Cognition Study - Demographics
Group 1 (n=30)
Age (years)
Gender
Race
Education
Marital Status
Single
Married
Divorced/Separated
Group 2 (n=30)
Mean
SD
Mean
46.9
10.38
45.93
SD
10.40
Male
Female
Male
Female
13
17
10
20
White
African
American
Other
White
African
American
Other
21
9
11
1
0
18
High School
University
or Less
Other
High School
University
or Less
Other
7
1
8
2
22
13
6
10
20
13
7
10
CONFIDENTIAL
24
Cognition study results
Effect Size - Sensoril 500 mg/day
Cohen’s “d” Effect Size
a
0.2
Small
0.5
Medium
0.8
Large
b
a
b
Working memory – Digit Span Backwards: (a) number correct backward; (b) span backward
Social Cognition - Penn Emotional Acuity Test: mean response rating; Attention - Flankers
Continuous Performance Test: (a) RT neutral mean; (b) RT neutral median
CONFIDENTIAL
25
Cognition study results –Adverse Events
Sensoril Study- Adverse Events by Event (n=60)
Nervous System

Sleepiness

headache

vivid
i id dreams/nightmares
d
/ i ht

dizziness

tiredness/fatigue

lightheadedness
Gastrointestinal

nausea/upset stomach

constipation

diarrhea

decrease in appetite

increase in gas
Dermatological

rash

itching
Cardiovascular

palpitations
Circulatory

swelling in feet

heaviness in legs

tingling in fingers
Psychiatric

increase in depression
Genitourinary

frequent urination
Respiratory
19 Events in Sensoril (n=30)
26 Events in Placebo (n=30)
3
0
3
1
1
0
6
2
3
0
2
1
1
0
5
0
1
4
2
1
1
0
1
0
1
1
0
1
1
0
0
0
1
1
1
1
1
0
CONFIDENTIAL
26
Effect size of Latuda® and Lexapro® from Literature:
Latuda
(Lurasidone)
(L
id
)
Lexapro
(Escitalopram)
40 mg/day
120 mg/day
10 mg/day
20 mg/day
NOTE: The data shown here is not cognition data and presented only
to give an idea about the effect sizes with psychiatry drugs
CONFIDENTIAL
27
Cognition Study - Summary

The effect sizes for working memory (digit span test), social
cognition (using the PENN emotional acuity test), or attention
(using the Continous Performance Test – Flankers version) were in
favor of Sensoril over placebo.

Medium to close to large effect sizes for cognition studies in
psychiatry are not at all common; thus for a 2 month study with
Sensoril® at a 500 mg/day the results are encouraging.

When add on drugs are administered along with CNS drugs, there
are often more side effects; however,
however when Sensoril® was given
along with other CNS drugs in this 2 month study, the side effect
profile was quite benign.

Study was done as per IND filed with US FDA and not funded by
Natreon.
CONFIDENTIAL
28
Effect of Sensoril® on Cold Pressor-induced
Cardiovascular Changes in Healthy Volunteers
Study Design: Randomized, double-blind, placebocontrolled, crossover
Subjects: 20 healthy male subjects
Dosage: 2 x Sensoril® 250mg BID
2 x Matching placebo capsules BID
Duration of treatment: 2 weeks
CONFIDENTIAL
29
Effect of Sensoril® on Cold Pressor-induced
Change in Aortic Pressure
Aortic Pressure, mmHg
Mean
n % Change
Mean % Change in Aortic Pressure Induced by Cold
Pressor Test with Sensoril® & Placebo
Data Expressed as Mean ± SD
CONFIDENTIAL
30
Effect of Sensoril® on Cold Pressor-induced
Change in Augmentation Index
Augme
entation Index
Mean
n % Change
Mean % Change in Augmentation Index Induced by
Cold Pressor Test with Sensoril® & Placebo
***
Data Expressed as Mean ± SD
***p<0.001 Compared to Pretreatment
CONFIDENTIAL
31
Effect of Sensoril® on Mental Stress-induced
Cardiovascular Changes in Healthy Volunteers
Study Design: Randomized, double-blind, placebocontrolled, crossover
Subjects: 20 healthy male subjects
Dosage: 2 x Sensoril® 250mg BID
2 x Matching placebo capsules BID
Duration of treatment: 2 weeks
CONFIDENTIAL
32
Effect of Sensoril® on Mental Stress-induced
Change in Aortic Pressure
Aortic
c Pressure, mmHg
Me
ean % Change
Mean % Change in Aortic Pressure Induced by
Mental Stress Test with Sensoril® & Placebo
*
Data Expressed as Mean ± SD
*p<0.05 Compared to Pretreatment
CONFIDENTIAL
33
Effect of Sensoril® on Mental Stress-induced
Change in Augmentation Index
Augm
mentation Index
Mean % Change
Mean % Change in Augmentation Index Induced by
Mental Stress Test with Sensoril® & Placebo
**
Data Expressed as Mean ± SD
**p<0.01 Compared to Pretreatment
CONFIDENTIAL
34
Effect of Sensoril® on Psychomotor
Performance in Healthy Volunteers
Study Design: Randomized, double-blind, placebocontrolled,
ll d crossover
Subjects: 20 healthy male subjects
Dosage: 2 x Sensoril® 250mg BID
2 x Matching placebo capsules BID
Duration of treatment: 2 weeks
CONFIDENTIAL
35
Effect of Sensoril® on Psychomotor
Performance in Healthy Volunteers
Test Reaction Time with Sensoril®, ms
Pre Treatment
Post Treatment
187±16
183±17 (ns)
306±39 287±43 ** Reaction Time with Placebo, ms Pre Treatment
Post Treatment 192±14
194±14 (ns) 313±45 316±26 (ns) Finger tapping Simple reaction test Choice discrimination test 471±34 455±51 * 476±34 Digit Symbol Substitution Test 1695±263 1557±233 ** 1690±279 Digit vigilance task 1360±57
1317±88 **
1371±79
Card sorting test 96±19 84±13 * 98±17 P value compared to baseline in Reaction Time
p
477±30 (ns) 1739±313 (ns) 1395±82 (ns) 98±20 (ns) ns – Not significant; * p<0.05; ** p<0.001 CONFIDENTIAL
36
Effect of Sensoril® on Pain Threshold Force and
Threshold Time in Healthy Volunteers
Mechanical Pain Model
Placebo
Sensoril®
p value
Baseline threshold force
(grams)
443.06 ±
65.02
450.3 ± 75.49
ns
Post Drug threshold force
(grams)
446.53 ±
65.95
538.5 ± 72.27
<0.05
Baseline threshold Time
(seconds)
5.54 ± 0.81
5.62 ± 0.94
ns
Post Drug threshold Time
(seconds)
5.58 ± 0.82
6.73 ± 0.90
<0.05
All values - Mean ± Standard deviation
p values represent the students paired t test between
the placebo and Sensoril® group
Effect of Sensoril® on Pain Threshold Time
Healthy Volunteers
Hot Air Pain Model
100
Time in seconds
80
p<0.001 $
60
p-ns*
baseline
postdrug
40
20
44.28 ± 9.19
43.79 ± 6.79
43.99 ± 6.93
49.89 ± 7.07
0
Placebo
Sensoril
Values given as Mean ± SD
* compared to baseline
$ compared to baseline and placebo
Sensoril® - Summary

Reduces stress, fatigue and sleeplessness and increases energy

Improves endothelial function and lipid profile in Type 2
diabetics

Improves mental cognition without significant adverse events

Decreases hsCRP, a biomarker for inflammation.

Backed by 7 clinical studies, self-affirmed GRAS, Pre-DSHEA

No complaints in 9 years of world-wide market history
CONFIDENTIAL
39
Ashwagnadha
Analytical Methodology
40
Current Specifications of Bioactives in
Sensoril® brand Ashwagandha

Withanolide glycosides (combined Withanoside Iv,
With
Withanoside
id V,
V Withanolide
With
lid A,
A etc.)
t ) : ≥ 10
10.0%
0%

Withanolide aglycones as Withaferin-A : ≤ 0.5%

Oligosaccharides : ≥ 32.0%
CONFIDENTIAL
41
Current HPLC Method - Quantification of
Withanolide Glycosides and Withaferin-A

HPLC Condtions:
Merck-Hiber® Pre-Packed column RT 250-4, LiChrosorb® RP-18, Particle
size 5μM, 4 x 250 mm cartridge column, Ord No.1.50333.0001 with a
reverse phase guard column.
A bi t
Ambient
Acetonitrile: Water-1: 1 (v/v)
0.6 ml/min
20 min
Isocratic
UV 225 nm
20μl (with a loop injector)
Waters HPLC 515 PDA Detector (Waters™ 2996, Photodiode Array
Detector), evaluation with Empower Software
Pdt. No. UN-1648 Acetonitrile (Merck), Pdt No. 93956 Water for HPLC
(Merck)
Method with external standard; evaluation of area of peaks.
Withaferin A isolated from Withania somnifera by multiple column
chromatography was used as external standard. Withaferin A, 0.5 mg was
dissolved in 1 ml of methanol and four different concentrations were
applied to HPLC. Calibration curve was plotted between area and different
concentration. The linear regression equation of the calibration curve was
obtained as follows:
Y = 2217082.726 X+ 200001.715 with a correlation coefficient of 0.9998.
Where Y is the area and X is the concentration in g.
CONFIDENTIAL
42
Current HPLC Method - Quantification of
Withanolide Glycosides and Withaferin-A



Withaferin-A reference standard, developed in-house, is used for
both aglycone as well as glycoside estimation
The peak appearing at tR 7.5 min is considered that of
With f i A
Withaferin-A
Peaks appearing at tR 2.0-7.4 min are considered those of
withanolide glycosides.
2.865
2 .4 0
2 .2 0
2 .0 0
1 .8 0
1 .6 0
4.854
0 .4 0
0 .2 0
10.339
0 .6 0
7.559
0 .8 0
5.668
1 .0 0
8.672
1 .2
20
Peak2 - 3.581
AU
U
1 .4 0
0 .0 0
2 .0 0
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
1 2 .0 0
1 4 .0 0
M in u te s
43
Current HPLC Method - Quantification of
Oligosaccharides

HPLC Conditions:
Equipment
Waters HPLC system consisting RI detector Waters 2414, pump Waters 515,
evaluation with Empower software.
Column
C
l
Column
Temperature
Eluent
C b h d t Analysis
Carbohydrate
A l i Column
C l
[ t
[waters]
] 300 x 3.9
3 9 mm, Part
P t No.WAT
N WAT 084038
Ambient
Acetonitrile: Water in the ratio of 80:20 (v/v)
Run Time
10 min
Flow rate
2 ml/min
Gradient
Isocratic
Programme
Injection volume
Sensitivity
Reagents
20μl (with a loop injector)
16
Pdt. No. UN-1648 Acetonitrile (Merck)
Pdt. No. 93956 Water for HPLC (Merck)
Evaluation
Method with external standard.
standard
Oligosaccharide isolated from Withania somnifera by multiple
column chromatographies was used as external standard.
Standard
Oligosaccharide 10 mg was weighed and dissolved in 1 ml of
water. Four different concentrations (10g, 50g, 100g and
200g in 20l) were applied to HPLC. Calibration curve was
plotted between concentration and area. The linear regression
equation was obtained as follows.
Y = 6620.5 X+ 72099 with a co-relation coefficient of 0.996
where Y is the area and X is the concentration in g.
The area of peaks appearing between 4-10 minutes is added
and contents of oligosaccharides are calculated using linear
regression equation
External
44
New HPLC Method

Reference standards of Glycosides (Withanoside
(Withanoside-IV,
IV,
Withanoside-V, Withanolide-A, Withanoside-UV active)
and Aglycones (Withaferin-A, Withastramonolide,
Withanone) were obtained from ChromaDex.

Withanoside IV is used to calculate the total withanolide
glycoside content

Withaferin-A is used to calculate the total aglycone content
CONFIDENTIAL
45
New HPLC Method – HPLC Conditions
HPLC conditions:
System
: Alliance-Water (PDA 2996)
System number
: NAT/HPLC/03
Method
y
: Sensoril Gradient system
Column
: Lichrocart (250 × 4 mm, 5 μm Merck) [NI/LC/RPC18/005[250×4]]
Solvent system
: Gradient; (A) Acetonitrile (B) Water
Detection
: 225 nm (PDA detector)
Flow rate
: 0.5 ml/min
Run time
: 30 min
HPLC programming for mobile phase is given below:
Table 1: Gradient system of solvent-A and solvent-B
Time (Min.) Solvent A (%) Solvent B (%)
0
10
90
4.0
20
80
7.0
80
20
10.0
70
30
15.0
60
40
17.0
100
0
22.0
100
0
25.0
20
80
30.0
10
90
46
New HPLC Method - Sensoril® HPLC
Chromatogram
47
New HPLC Method – Retention Times of
Standards
Sample
Retention Time (Min.)
Withanoside-IV
11.20
Withanoside-V
11.96
Withanoside-UV-active
11.23
Withaferin-A
13.08
Withanone
14.42
Withastramonolide
13.64
48
New HPLC Method – Calibration Curve for
Withanoside IV
49
New HPLC Method – Calibration Curve for
Withaferin-A
50
Current HPLC Method vs New HPLC Method
Sensoril Monograph Method
Batches
New HPLC Method
Glycoside
Aglycone
Glycoside
(% w/w)
(% w/w)
(% w/w)
(% w/w)
WS1307165
10.49
0.22
13.77
0.53
WS1311173
9.46
0.214
15.13
0.73
WS1311174
10.42
0.218
21.90
1.01
WS1311175
12.54
0.29
17.53
0.79
WS1311176
12.61
0.2
16.73
0.72
Aglycone
51