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A lupin seed γ-conglutin-enriched preparation was tested in a glucose overload trial with both murine models and adult healthy volunteers. The results with rats showed a dose-dependent significant decrease of blood glucose concentration, which confirmed previous findings obtained with the purified protein. Moreover, three test-product doses equivalent to 630, 315, and 157.5 mg γ-conglutin, orally administered 30 min before the carbohydrate supply, showed a relevant hypoglycemic effect in human trials. Insulin concentrations were not significantly affected. The general hematic parameters did not change at all. This is the first report on the glucose-lowering effect of lupin γ-conglutin in human subjects.
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1Hypoglycemic effect of lupin seed γ-conglutin in experimental animals and
2healthy human subjects
3Juan C. Bertoglio
, Mario A. Calvo
, Juan L. Hancke
, Rafael A. Burgos
, Antonella Riva
4Paolo Morazzoni
, Cesare Ponzone
, Chiara Magni
, Marcello Duranti
Instituto de Medicina, Valdivia, Chile
Instituto de Farmacologia Universidad Austral de Chile,Valdivia, Chile
Indena S.p.A., Milan, Italy
Dept. of AgriFood Molecular Sciences, Università degli Studi di Milano, Italy
11 article info12 abstract
13 Article history:
14 Received 18 March 2011
15 Received in revised form 6 May 2011
16 Accepted 8 May 2011
17 Available online xxxx
18 A lupin seed γ-conglutin-enriched preparation was tested in a glucose overload trial with both
19 murine models and adult healthy volunteers. The results with rats showed a dose-dependent
20 significant decrease of blood glucose concentration, which confirmed previous findings
21 obtained with the purified protein. Moreover, three test-product doses equivalent to 630, 315,
22 and 157.5 mg γ-conglutin, orally administered 30 min before the carbohydrate supply, showed
23 a relevant hypoglycemic effect in human trials. Insulin concentrations were not signicantly
24 affected. The general hematic parameters did not change at all.
25 This is the first report on the glucose-lowering effect of lupin γ-conglutin in human subjects.
26 © 2011 Published by Elsevier B.V.
27 Keywords:
28 Antidiabetic
29 Lupinus albus
30 Protein extract
31 Animal trial
32 Human study3334
36 Introduction
37 Hyperglycemia is recognized to be the central feature of all
38 unbalances in the metabolism of carbohydrates, lipids,
39 ketones and amino acids [1]. The most diffused pathological
40 condition, characterized by stable hyperglycemia, is known
41 today as type-2 diabetes (accounting for about 90% of all
42 diabetes cases). Nowadays, type-2 diabetes is considered an
43 epidemic disease, especially in the Western countries, where
44 the incidence in the population is estimated to range from 2%
45 up to 4% [2]. Type-2 diabetes is usually preceded by years of
46 an abnormal condition, termed impaired glucose tolerance
47 (IGT) characterized by plasma glucose levels between 140
48and 199 mg/dL, 2 hours after a standard oral glucose
49challenge, but not as high as in diabetes (N200 mg/dL).
50Several characteristics in the population have been recog-
51nized to be associated with a greater risk of progression from
52IGT to type 2 diabetes. Among these are impaired insulin
53secretion, insulin resistance, obesity and age Q1[2-4]. Therefore,
54actions aimed at controlling this situation are crucial.
55Lupin is a leguminous seed which has largely been used as
56a food for its high protein content in the Mediterranean area
57since thousand years. Around the year 1000 AD, the Persian
58doctor Ibn Sīnā,amongthersts to describe diabetes
59symptoms, used mixtures of lupin, fenugreek and zedoary
60seed ours to remarkably reduce sugar excretion. Lupin seed
61is mentioned in the ancient and traditional pharmacopoeia
62books as an anti-diabetic product. Last century, in the search
63of the lupin seed active principle, Orestano described the
64extraction and purication from white lupin seeds of a
65compound capable of decreasing glycemia in rabbits. How-
66ever, the extraction was tedious and the yield extremely low
67and thus the product was considered to be lacking of any
Fitoterapia xxx (2011) xxxxxx
Abbreviations: BMI, body mass index; BUN, blood urea nitrogen; GOT,
glutamate-oxaloacetate transaminase; GPT, glutamate-pyruvate transami-
nase; GGT, γ-glutamyl transpeptidase; LDH, lactate dehydrogenase.
Corresponding author at: Department of AgriFood Molecular Sciences,
Università degli Studi di Milano, Via Celoria, 2 I-20133 Milano, Italy.
Tel.: +39 02 503 16817; fax: + 39 02 503 16801.
E-mail address: (M. Duranti).
FITOTE-02218; No of Pages 6
0367-326X/$ see front matter © 2011 Published by Elsevier B.V.
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Please cite this article as: Bertoglio JC, et al., Hypoglycemic effect of lupin seed γ-conglutin in experimental animals and
healthy human subjects, Fitoterapia (2011), doi:10.1016/j.tote.2011.05.007
68 potential application [5]. The possibility that a lupin seed
69 protein could be the active principle was not taken in
70 consideration until recently.
71 During our research activities on lupin seed proteins, we
72 undertook a series of research activities aimed at unraveling the
73 specic hypoglycemic role of one of the lupin proteins, termed γ-
74 conglutin. Lupin γ-conglutin is a homo-tetrameric glycoprotein
75 in which the monomeric unit consists of two disulphide linked
76 heterogenous subunits of about 30 and 17 kDa (for a review on
77 γ-conglutin properties, see reference 6). From the viewpoint of
78 γ-conglutin biological activity, orally-administered pure γ-
79 conglutin was found to effectively decrease plasma glucose in
80 glucose overloaded rats in a dose-dependent manner [7].More
81 recently, the insulin-mimetic activity of γ-conglutin on differen-
82 tiating myoblasts was described [8].
83 In this work, the remarkable glucose lowering capacity of
84 a lupin γ-conglutin-enriched preparation was conrmed in
85 rat models and assessed for the rst time in healthy human
86 subjects.
87 2. Materials and methods
88 2.1. Lupin seed γ-conglutin laboratory purication
89 Lupinus albus L. seed γ-conglutin was puried to homogene-
90 ity from dehulled dry lupin seeds by using a combination of
91 chromatographic steps, as described by Duranti et al. [9].
92 2.2. Lupin seed γ-conglutin preparation for animal and human
93 studies
94 A lupin γ-conglutin-enriched dry extract (Pro-Gamma
95 was prepared by INDENA S.p.A., Milano, Italy, according to an
96 industrially-developed procedure (WO 2004/071521) which
97 consisted essentially in a protein wet extraction process and
98 solvent precipitation. The test product for the study was
99 manufactured in the form of dried powder. The protein
100 content of the dry powder, as assessed by the Lowry method
101 [10], was 44.8% of the dry weight. γ-Conglutin content,
102 evaluated by SDS-PAGE densitometric scanning, was about
103 47% of the total proteins in the dry powder (see below).
104 The test product was used as such in rat studies at the
105 dosages of 50, 100 and 200 mg/kg b.w. (see below details on
106 the administration procedure). As far as the human trials are
107 concerned, the dry powder was packed in vacuum sealed
108 sachets, for easy handling, containing 1500 mg powder each
109 (equivalent to 315 mg of γ-conglutin) by FARMINDUSTRIA
110 S.A. Laboratories (Santiago, Chile). For placebo, the formula
111 was the same as for the verum formulation, except for the
112 exclusion of lupin dry extract, which was substituted by
113 microcrystalline cellulose (Avicel PH302). The test product
114 and the placebo were kept at room temperature in a dry place
115 and protected from light.
116 2.3. SDS-PAGE
117 SDS-PAGE was carried out on 12% polyacrylamide gels,
118 according to Laemmli [11] under reducing and non-reducing
119 conditions using a mini-PROTEAN II cell (Bio-Rad). The gels
120 were Coomassie blue stained.
121The SDS-PAGE images were acquired using a scanner,
122Canoscan 8000F (Canon, Milan, Italy) interfaced with a
123personal computer and densitometric scans were carried
124out with ImageMaster 1D software (Amersham Pharmacia
125Biotech, Milan, Italy).
1262.4. Design of the animal study
127A total of 100 male rats (Charles River, Calco, LC, Italy)
128with an average body weight ranging between 275 and 300 g
129were maintained under stable conditions for 7 days before
130the experiment. The animals were given a standard rat diet
131and were kept under automatically controlled light, temper-
132ature, and humidity conditions. The rats were divided into
133ve groups. One group, the control group, received only the
134control product, i.e., the placebo; three groups received 50,
135100, or 200 mg/kg body weight of the γ-conglutin-enriched
136test product, corresponding to 10.5, 21.0 and 42.0 mg γ-
137conglutin; the last group was given 50 mg/kg body weight of
138metformin added to the control product. Administration was
139carried out by gavage, 30 min before the glucose overloading
140experiment. At time 0 of the experiment, each rat was given
1412 g/kg body weight glucose administered orally. At estab-
142lished times thereafter (0, 30, 60 and 90 min from glucose
143administration), each rat in a group of 5 rats per time and
144dose was treated with 50 mg/kg body weight Na-thiopenthal,
145and 5 mL of blood were collected in 7.5 mmol/L EDTA
146containing tubes. The blood was immediately centrifuged at
1472000×gat 4 °C for 10 min and the supernatant used for
148glucose assays. All procedures involving rats and their care
149were performed according to the Italian Government Guide-
150lines for animal tests and were in agreement with the
151European Commission rules (86/609/EEC).
1522.5. Design of the human trial
153The present trial was a placebo-controlled study con-
154ducted on fteen adult healthy volunteers, who received
155three different single test doses of respectively 750 (half the
156content of 1 sachet), 1500 (1 sachet) and 3000 mg (2 sachet)
157of the test product, corresponding to 157.5, 315 and 630 mg
158γ-conglutin, respectively, and a placebo, with no γ-conglutin
159added. The whole duration of the trial was 7 weeks. The three
160doses and the placebo were administered per os 30 min
161before a carbohydrate meal, consisting in one serving of 85 g
162boiled (Grade 1) white rice which corresponded to an intake
163of 75 g carbohydrate. The owchart of the clinical study is
164reported in Table 1.
165The study was organized and directed by Patagonia
166Clinical Trials, CRO program of the Institute of Pharmacology,
167Universidad Austral de Chile in the city of Valdivia, Chile. All
168the volunteers were recruited from the city of Valdivia. The
169study started after approval by the local Bioethical Committee
170with the recruitment of the volunteers and was completed
171within 5 weeks from the beginning of the rst session.
172Written informed consent was obtained from all subjects.
1732.6. Recruitment of the subjects
174Fifteen healthy adult (N18 years old) male and female
175volunteers, who complied with the inclusion criteria, were
2J.C. Bertoglio et al. / Fitoterapia xxx (2011) xxxxxx
Please cite this article as: Bertoglio JC, et al., Hypoglycemic effect of lupin seed γ-conglutin in experimental animals and
healthy human subjects, Fitoterapia (2011), doi:10.1016/j.tote.2011.05.007
176 selected in November 2008. The selected adult volunteers were
177 not receiving any acute or chronicpharmacological therapy and
178 had a body mass index (BMI) less than 30 kg/m
. All subjects
179 who were enrolled for the present study had a fasting plasma
180 glucose (BG) b100 mg/100 mL (or b5.6 mmol/L), and a normal
181 oral glucose tolerance test (OGTT), dened as a plasma glucose
182 between 100 mg/100 mL and 140 mg/100 mL (or 5.6 and
183 7.8 mmol/L, respectively), at 120 min after a 75 g serving of
184 carbohydrates [2,4,12].
185 2.7. Exclusion criteria
186 The following criteria were used to exclude candidate
187 volunteers:
188 elevation of glycemia in plasma up to 140 mg/dL or
189 above, after 75 g of carbohydrate load;
190 diabetes mellitus;
191 hospitalization over the last 60 days;
192 arrhythmia.
193 renal failure (blood creatinine N1.5 mg/dL);
194 documented allergy to peanuts;
195 any other chronic or limiting disease, including
196 alcoholism;
197 any chronic pharmacological treatment;
198 any disease or condition that the enrolling physician
199 considered making the subject unsuitable for participation
200 to this trial;
201 pregnant women and women under hormonal contra-
202 ception method prescribed less than 2 months before.
203 2.8. Statistical analysis
204 Results were depicted as the mean ±SME. The incremen-
205 tal area under the glucose and insulin curves were calculated
206 as described by Wolever and Jenkins [13]. The statistical
207 signicance was calculated using the paired t-test and a
208 statistical signicance of Pb0.05 was adopted.
209 In addition, an exploratory analysis for all dataset and
210 separately for each treatment group was also performed.
211 Descriptive statistics and 95% condence intervals were used
212 to estimate parameters. To evaluate normality assumption we
213 used ShapiroWilks test, considering non-normal distribution
214 those presenting p-values lower than 0.05. To evaluate the
215 degree of independence between repeated measures during
216 the period of study, the Pearson correlation test was used. A
217moderate to high intra-individual correlation to all outcome
218variables over repeated measurements was observed (data not
219shown),therefore a generalizedestimating equation(GEE) was
220used to evaluate these data, considering an exchangeable
221correlation structure by robust standard errors, using the
222identity link function which works with normal distribution
223[14-17]. The outcome variables (glucose and insulin) were
224modeled considering treatment variable [(0=placebo; dose 1
225(750 mg), 2 (1500 mg) and 3 (3000 mg)] plus interaction
226variable (group*time). This model was adjusted by body max
227index (BMI). In all adjusted models, the interaction terms were
228non-signicant, indicating that the treatment effect on the
229outcome does not vary signicantly over the time among
230groups (data not shown). The xtgee procedure of STATA was
231used to evaluate these models [18]. All analyses were done
232considering on a base of intention to treat. Graphical methods
233were used to show glucose and insulin changes over time
234(minutes). In all models, timevariable was used as a continuous
2363. Results and discussion
2373.1. Characterization of γ-conglutin preparations by SDS-PAGE
238The electrophoretic pattern under reducing and non-
239reducing conditions of the sample product used in this work
240for the animal and human studies (Fig. 1, lanes 2) is compared
241with the placebo preparation, which did not contain γ-
242conglutin (Fig. 1, lanes 1) and a laboratory puried γ-conglutin
243control (Fig. 1, lanes 3). The characteristic main band of γ-
244conglutin under non-reducing conditions with anM
of about
24550 kDa was visible in the panel B, lanes 2 and 3. When reducing
246conditions were applied, the50 kDa polypeptide of γ-conglutin
247underwent reduction giving rise to the 30 and 17 kDa subunits
248of this lupin protein [6], which can be seen in the panel A of the
250As far as purity of the sample is concerned, a number of
251minor bands were visible in the industrial γ-conglutin
252preparation, but not in the laboratory puried protein, as
253expected. Therefore, the relative amounts of γ-conglutin
254polypeptides in the industrial sample were quantied by
255densitometric scanning of both reduced and non-reduced
256samples in order to assess the actual γ-conglutin amounts in
257the formulates. With the reduced sample, 46.9± 5.0% γ-
258conglutin with respect to the total polypeptides was obtained
259in triplicate analyses, while 60.0 ± 7.0% γ-conglutin was
260obtained with the non-reduced sample. This difference can
Table 1t1:1
Human trial owchart of each experimental session.
t1:3Time (minutes) BMI Plasma glucose and insulin Creatinine, BUN, total cholesterol, triglycerides,
GOT, GPT, GGT, bilirubin, alkaline phosphatase, LDH.
t1:410 (Baseline) Yes Yes In sessions: placebo, 750 and 3000 mg, only
t1:50 (Test product) No No No
t1:615 No Yes No
t1:730 (Carbohydrate intake) No Yes No
t1:860 No Yes No
t1:990 No Yes No
t1:10 120 No Yes No
t1:11 180 No Yes In sessions: placebo, 750 and 3000 mg, only
3J.C. Bertoglio et al. / Fitoterapia xxx (2011) xxxxxx
Please cite this article as: Bertoglio JC, et al., Hypoglycemic effect of lupin seed γ-conglutin in experimental animals and
healthy human subjects, Fitoterapia (2011), doi:10.1016/j.tote.2011.05.007
261 be explained with the presence of insoluble protein material
262 in the non-reduced sample, which was removed before the
263 electrophoretic run or did not enter the gel network. On this
264 basis, the lower γ-conglutin estimate, i.e., 46.9% of the total
265 proteins, and the protein content of the dry powder, i.e.,
266 44.8%, were both considered for the quantication of γ-
267 conglutin as the active principle concentration in the
268 formulates. According to this evaluation, the amounts of γ-
269 conglutin in the three administered dosages of formulates
270 used for human studies, i.e., 750, 1500 and 3000 mg, were
271 157.5, 315 and 630 mg, respectively.
272 3.2. Results of the animal study
273 The γ-conglutin-enriched preparation was orally admin-
274 istered to rats prior to glucose overload and blood glucose
275 concentrations were monitored for 90 min. The whole
276 procedure is detailed under Materials and methods. The
277 administration of 2 g/kg glucose caused a fourfold increase of
278 blood glucose in control rats from 70 ±9 to 285 ±28 mg/dL;
279 Pb0.001. The greatest increase occurred after 30 min from
280 glucose intake and then, as physiologically expected, concen-
281 trations decreased (not shown). Rat pre-treatment with
282 the test product administered prior to glucose overload at
283 the dosages of 50, 100 and 200 mg/kg b.w (corresponding
284 to 10.5, 21.0 and 42.0 mg γ-conglutin) decreased the
285 blood glucose increase in a dose-dependent pattern. As it
286 can be seen in Fig. 2, the areas under the curve (AUC) of
287 glucose concentrations decreased by 14% (PN0.05), 42%
288 (Pb0.01) and 64% (Pb0.001) at the three mentioned doses,
289 respectively, in comparison to the control treatment. The
290 effect obtained with the highest dose did not signicantly
291 differ from the action of metformin at 50 mg/kg b.w. This
292 nding conrms previous results obtained with pure γ-
293 conglutin [7], where the hypoglycemic effect of the lupin
294 protein was rst described.
2953.3. Results of the clinical study
296The tolerability of the L. albus puried dry extract was fully
297satisfactory. No clinical subjective side effects were observed,
298nor any adverse events recorded upon administration and
299during the time course of this study. All vital signs remained
300unwavering, including appetite and uid intake. Hemody-
301namic parameters were stable; clinical laboratory analysis of
302blood chemistry concerning creatinine, urea nitrogen (BUN),
303total cholesterol, triglycerides, glutamate-oxalacetate trans-
304aminase (GOT), glutamate-pyruvate transaminase (GPT),
305gamma-glutamil transpeptidase (GGT), bilirubin, alkaline
306phosphatase and lactate dehydrogenase (LDH), also
307remained unchanged and stable throughout (not shown).
308The glucose and insulin plasma levels (means ±SME)
309during the time course of the clinical study are shown in
11 2 23 3 2 21 1
Fig. 1. SDS-PAGE under reducing (A) and non-reducing conditions (B) of γ-conglutin preparations. The gels were stained by Coomassie brilliant blue. The lanes
with same numbers refer to two differently loaded volumes of the same sample. Sample lanes are the following: 1: placebo preparations without γ-conglutin; 2:
industrial scale γ-conglutin preparation; 3: laboratory scale γ-conglutin preparation (4 μg protein).
Dosage groups (mg test product/kg b.w.)
AUC (mg / dL x min90)
Control 50 100 200 Metformin
*** ***
Fig. 2. Histogram of the areas under the curve, AUC, of plasma glucose
concentrations in the rat study during the 90 min trial at the three doses
indicated in the gure. No γ-conglutin was administered in the control group,
while metformin at 50 mg/kg b.w. was used as a positive control.The data are
means± SEM of ve animals in each dosagegroup at 0, 30, 60 and 90 min from
glucose overload. *Pb0.05, **Pb0.01 and ***Pb0.001 vs. the control.
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Please cite this article as: Bertoglio JC, et al., Hypoglycemic effect of lupin seed γ-conglutin in experimental animals and
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310 Fig. 3, panels A and B, respectively. The changes of glucose
311 concentrations in the plasma of all fteen subjects at the three
312 doses tested, with respect to placebo, were remarkable. In
313 particular, statistically signicant and comparable reduction
314 with the three doses of formulate was observed at 60 min,
315 where the average glucose concentration was around 81% of
316 the placebo control. At 90 min, the shapes of the curves for
317 the three doses differentiated, being the lowest dose more
318 similar to the placebo control (83%). Conversely, the
319 intermediate dose, 1500 mg, had the greatest effect (61% of
320 the placebo) and the greatest dose, 3000 mg, resulted in 71%
321 of the placebo. At 120 min, the highest doses still maintained
322 a statistically signicant reduction, being the 71% of the
323 placebo control. The overall shapes of the highest doses
324 curves were very similar, thus suggesting that the 1500 mg
325 dose already reached its maximum effect.
326 In parallel, insulin plasma concentrations of all fteen
327 subjects, shown in panel B of Fig. 3, showed an increase in the
328 absolute amount of secreted insulin at 60 min at all doses,
329then a relatively homogenous pattern of decay followed, with
330the data not signicantly differing from the placebo.
331Therefore, the main outcome of these results was the
332efcacy of γ-conglutin oral administration on the modica-
333tion of post-prandial plasma glucose, while the overall
334pattern of insulin response was less affected. The hypoglyce-
335mic effect of laboratory-puried homogenous γ-conglutin
336had been already observed in glucose overloaded animal
337models [7] and its bioactivity was demonstrated in cell
338models too [8]. Therefore, the identication of γ-conglutin as
339the active principle is not under discussion in this work.
340Data are also reported as variations of the areas under the
341curve (AUC) in Fig. 4, panels A and B for glucose and insulin,
342respectively. Glucose concentration decreased in a statistically
343signicant manner at the two highest doses of γ-conglutin, 75%
344and 79% of the placebo for 1500 mg and 3000 mg, respectively.
345The lowest dose also showed a decreasing trend, but its
346difference with the placebo was not statistically signicant. As
347far as insulin plasma levels are concerned, no statistically
348signicant difference was observed upon the treatments.
349The subsequent statistical analysis by means of the GEE
350(adjusting glucose and insulin plasma levels for BMI and time)
351is reported in Table 2. The results showed that treatment vs.
352placebo variable did havea negative signicant association only
030 60 90 120 150 180
1,500 mg 3,000 mg
750 mg
time (min)
Glycaemia (mg / dL)
1,500 mg 3,000 mg
750 mg
Insulin (µlU / mL)
0 30 60 90 120 150 180
time (min)
Fig. 3. Time course of plasma glucose (panel A) and insulin (panel B)
concentrations in healthy volunteers (n= 15) treated with the test product
and a placebo, after starch intake.
0 (Placebo) 750 1,500
** *
Lupin dosage groups (mg)
AUC (mg / dL x min180)
0 (Placebo) 750 1,500 3,000
Lupin dosage groups (mg)
AUC (µIU / mL x min180)
Fig. 4. Histogramof the areas under thecurve, AUC, of plasma glucose(panel A)
and insulin concentrations (panel B) in the human study during the 180 min
trial at the threedoses indicated in the gure.No γ-conglutin was administered
in the control group (placebo). Thedata are means ±SEM of 15 blood samples.
*Pb0.05, **Pb0.01 vs. the control.
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Please cite this article as: Bertoglio JC, et al., Hypoglycemic effect of lupin seed γ-conglutin in experimental animals and
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353 with blood glucose levels. The three dosages used showed an
354 average decrease of 8.71 (12.42 to 4.99; Pb0.001),
355 11.35 (17.23 to 5.46; Pb0.002) and 8.98 (14.53 to
356 3.43; Pb0.001) mg/dL, respectively. Conversely, no signi-
357 cant effects on insulin plasma levels were observed.
358 Altogether, these results support the conclusion that γ-
359 conglutin does not affect insulin secretion, rather, by
360 decreasing glucose concentrations, plays a role as a insulin-
361 mimetic compound, as already suggested in a previous work
362 with cell models. Indeed, the treatment of myocites with γ-
363 conglutin was found to activate several proteins and enzymes
364 involved in the insulin signaling pathway, to trigger protein
365 biosynthetic processes and induce differentiating mecha-
366 nisms closely resembling those monitored with similar
367 insulin concentrations [8].
368 4. Conclusions
369 In this study a γ-conglutin-enriched preparation was orally
370 administered to rats and found to be as effective as in a previous
371 work [7], where a homogenous γ-conglutin laboratory prepara-
372 tion was analyzed, thus conrming the biological activity of this
373 lupin protein as a glucose-lowering agent. Moreover, in the
374 present work the same test product, orally administered to
375 healthy adults with good tolerability, proved to induce a
376 signicant reduction of post-prandial plasma glucose concentra-
377 tion upon a standard load of carbohydrates, without a quantita-
378 tively signicant modication of the insulin secretion response.
379 Even the lowest product dose of 750 mg (equivalent to 157.5 mg
380 γ-conglutin), signicantly reduced the plasma glucose level
381 when adjusted for BMI. This seems particularly relevant in the
382 perspectives of using the extract in overweight subjects.
383 Several aspects, especially those related to γ-conglutin
384 metabolic fate and mechanism of action, still remain to be
385 investigated. Interestingly, recent data reporting stability of γ-
386 conglutin to digestive proteolytic attack, unless the protein is
387 completely denatured [19] would support the oral utilization of
388 this protein. Moreover, the possibility of transit of the lupin
389 protein through Caco-2 cell monolayers and in ex vivo models of
390 everted intestinal sacs has very recently been demonstrated [20].
391 With this work we have demonstrated for the rst time that
392 the active protein responsible for the claimed anti-diabetic
393 effect of the lupin seed is effective in man, in addition to animal
394 models. Further studies are needed for the best exploitation of
395 this dietary protein in the food and nutraceutical areas.
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Table 2t2:1
GEE analysis on human plasma glucose and insulin values adjusted by time and body mass index (BMI).
CI 95% p-
t2:4Lower Upper
t2:5Glucose (mg/dL) 750 mg 8.71 1.89 12.42 4.99 b0.001
t2:61500 mg 11.35 3.00 17.23 5.46 b0.002
t2:73000 mg 8.98 2.83 14.53 3.43 b0.001
t2:8Insulin (μIU/mL) 750 mg 1.13 0.93 0.69 2.94 0.224
t2:91500 mg 0.71 1.50 2.24 3.66 0.635
t2:10 3000 mg 0.99 1.17 1.31 3.28 0.400
6J.C. Bertoglio et al. / Fitoterapia xxx (2011) xxxxxx
Please cite this article as: Bertoglio JC, et al., Hypoglycemic effect of lupin seed γ-conglutin in experimental animals and
healthy human subjects, Fitoterapia (2011), doi:10.1016/j.tote.2011.05.007
... The treatment also resulted in a significant reduction of phytohemagglutinin-P (PHA) stimulated production of Th1 pro-inflammatory cytokines IL-2, IFN-γ, and TNF in human peripheral blood mononuclear cells (PBMCs) as well as a significant increase in TAC and ORAC antioxidant capacity of PBMCs of participants [116]. Bertoglio et al. (2011) investigated the effect of various doses (157.5, 315 and 630 mg) of lupin γ-conglutin ingested 30 min prior to a high carbohydrate meal on blood glucose and insulin responses of 15 healthy volunteers over three hours following the meal. A significant reduction of 25% and 21% in blood glucose level calculated as AUC was observed at the intermediate and the highest γ-conglutin doses, respectively. ...
... A significant reduction of 25% and 21% in blood glucose level calculated as AUC was observed at the intermediate and the highest γ-conglutin doses, respectively. There was no effect on insulin secretion and the authors concluded that γ-conglutin acts as an insulin mimetic based on an earlier cell model studies which showed that the treatment of myocytes with γ-conglutin activates proteins and enzymes in the insulin signaling pathway [117]. ...
... Compared to animal protein, legume consumption modulated inflammatory markers including a reduction in CRP, IL6, and TNF-α [116,120]. The high arginine (Arg) content of lupin protein and the specific lupin protein γconglutin are suggested to contribute to the favourable changes in glycemic control and reduced LDL cholesterol, and improved LDL:HDL ratios, especially in hypercholesterolemic individuals following lupin consumption [117]. In support of these changes, one study showed that lupin reduced the plasma concentrations of PCSK9, an important enzyme involved in the regulation of lipid metabolism and cholesterol reduction [115]. ...
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Future food security for healthy populations requires the development of safe, sustainably-produced protein foods to complement traditional dietary protein sources. To meet this need, a broad range of non-traditional protein foods are under active investigation. The aim of this review was to evaluate their potential effects on human health and to identify knowledge gaps, potential risks, and research opportunities. Non-traditional protein sources included are algae, cereals/grains, fresh fruit and vegetables, insects, mycoprotein, nuts, oil seeds, and legumes. Human, animal, and in vitro data suggest that non-traditional protein foods have compelling beneficial effects on human health, complementing traditional proteins (meat/poultry, soy, eggs, dairy). Improvements in cardiovascular health, lipid metabolism, muscle synthesis, and glycaemic control were the most frequently reported improvements in health-related endpoints. The mechanisms of benefit may arise from their diverse range of minerals, macro- and micronutrients, dietary fibre, and bioactive factors. Many were also reported to have anti-inflammatory, antihypertensive, and antioxidant activity. Across all protein sources examined, there is a strong need for quality human data from randomized controlled intervention studies. Opportunity lies in further understanding the potential effects of non-traditional proteins on the gut microbiome, immunity, inflammatory conditions, DNA damage, cognition, and cellular ageing. Safety, sustainability, and evidence-based health research will be vital to the development of high-quality complementary protein foods that enhance human health at all life stages.
... Lupines have gained increasing importance because of the beneficial health effects of their components (Mane et al. 2018). The Cc protein has obtained special interest due its hypoglycaemic activity, which has been already demonstrated in vitro, in vivo, and in human participants in clinical trials (Magni et al. 2004;Bertoglio et al. 2011;Lovati et al. 2012;Vargas-Guerrero et al. 2014;Mane et al. 2018). Although, the effects of lupine Cc on several molecules involved in the glucose metabolism have been evaluated (Vargas-Guerrero et al. 2014;Gonz alez-Santiago et al. 2017;Muñoz et al. 2018;Sandoval-Muñ ız et al. 2018), its full mechanism of action remains unknown. ...
... A protein band of $49 kDa was obtained under native conditions while two bands were observed under reducing conditions (17 and 29 kDa), which corresponded to the previously reported subunits of Cc (Duranti et al. 2008). These results are in agreement with those reported for Cc from domesticated lupine species isolated by different extraction methods (Bertoglio et al. 2011;Lovati et al. 2012;Vargas-Guerrero et al. 2014), and indicate that the protein extracted from L. rotundiflorus seeds corresponds to Cc. Differences in Cc lupine species have emphasized the importance of verifying the biological effects of Cc among species (Foley et al. 2015;Mane et al. 2018). In previous studies, Cc extracted from L. albus (domesticated species) was evaluated using the same treatment period (one week) and dose (120 mg/ kg/day) employed in the present study, and a reduction of 17-27% in serum glucose levels was reported (Vargas-Guerrero et al. 2014;Gonz alez-Santiago et al. 2017;Sandoval-Muñ ız et al. 2018). ...
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Context Gamma conglutin (Cγ) from lupine species represents a potential complementary treatment for type 2 diabetes mellitus (T2DM) because of its hypoglycaemic effect. However, its underlying mechanism of action is not fully known. Objective To evaluate whether Cγ from Lupinus rotundiflorus M. E. Jones (Fabaceae) modulates c-Jun N-terminal kinase 1 (JNK1) expression and activation in a T2DM rat model. Materials and methods Gamma conglutin isolated from L. rotundiflorus seeds was characterized by SDS-PAGE. Fifteen Wistar rats with streptozotocin-induced T2DM (HG) were randomized into three groups (n = 5): vehicle administration (HG-Ctrl), oral treatment with Cγ (120 mg/kg/day) (HG-Lr) for one week, and treatment with metformin (300 mg/kg/day) (HG-Met); a healthy group (Ctrl, n = 5) was included as control. The levels of glucose and biomarkers of renal and hepatic function were measured pre- and post-treatment. Hepatic Jnk1 expression and phosphorylation of JNK1 were evaluated by qRT-PCR and western blot, respectively. Results Oral treatment with either Cγ or metformin reduced serum glucose level to 86.30 and 74.80 mg/dL, respectively (p ˂ 0.05), from the basal levels. Jnk1 expression was 0.65- and 0.54-fold lower (p ˂ 0.05) in the HG-Lr and HG-Met groups, respectively, than in HG-Ctrl. Treatment with Cγ decreased JNK1 phosphorylation. However, Cγ did not change the levels of kidney and liver biomarkers. Discussion and conclusions Treatment with Cγ from L. rotundiflorus inhibited Jnk1 expression, in vivo, suggesting JNK1 as a potential therapeutic target in diabetes and revealing one mechanism underlying the hypoglycaemic effect of lupine Cγ. Nevertheless, further studies are required.
... (2011) conducted a placebo-controlled four-week trial that demonstrated that γ-conglutin administered before carbohydrate consumption exerted a hypoglycemic effect in healthy adults despite no significant variations in the observed insulin levels [146]. In addition, the addition of γ-conglutin to a sugary beverage was reported to acutely reduce glycaemia in type 2 diabetic individuals, suggesting that lupin protein could be a valuable tool in glycemic management [147]. ...
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Pulses and whole grains are considered staple foods that provide a significant amount of calories, fibre and protein, making them key food sources in a nutritionally balanced diet. Additionally , pulses and whole grains contain many bioactive compounds such as dietary fibre, resistant starch, phenolic compounds and mono-and polyunsaturated fatty acids that are known to combat chronic disease. Notably, recent research has demonstrated that protein derived from pulse and whole grain sources contains bioactive peptides that also possess disease-fighting properties. Mechanisms of action include inhibition or alteration of enzyme activities, vasodilatation, modulation of lipid metabolism and gut microbiome and oxidative stress reduction. Consumer demand for plant-based proteins has skyrocketed primarily based on the perceived health benefits and lower carbon footprint of consuming foods from plant sources versus animal. Therefore, more research should be invested in discovering the health-promoting effects that pulse and whole grain proteins have to offer.
... Furthermore, isolated lupin proteins of have been reported to have hyperlipidemic, anti-atherogenic, and hypocholesterolemic effects in rabbits, rats, and chickens [118,119]. Several clinical human studies have shown that lupin protein decreases total and LDL cholesterol, as well as triglyceride and reduce the glycaemic response ( Table 5) [120][121][122][123][124][125][126][127]. ...
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There is currently a trend in Western countries to increase the intake of plant proteins. In this chapter, the author explains that this is due to the beneficial physiological functions of plant proteins, based on the latest literature review and our own research results. Among plant proteins, soy protein has been reported to have many beneficial effects on the improvement and prevention of metabolic syndrome. This chapter outlines the excellent effects of soy protein on renal function [improvement of early symptoms of diabetic nephropathy], which is closely related to metabolic syndrome, and the effects of combining these effects as complementary medicine. In addition, recent findings about the anti-inflammatory and immune activation effects of soy protein as hydrolyzed peptides are outlined. A brief introduction of the recent results of other legume-derived proteins that have replaced soy proteins are also explained. By further deepening our understanding of the superior physiological functions of plant proteins, it is hoped that their use expands even further.
... Among the Lupinus spp., L. albus (white lupin), L. angustifolius (narrow-leaf lupin), and L. luteus (annual-yellow lupin) are the most consumed [4,5]. Several health-promoting properties have been reported of Lupinus species, mainly L. albus and L. angustifolius, such as antioxidant, anti-inflammatory, hypolipidemic, hypoglycemic, and hypotensive properties among others in several preclinical and clinical human and animal studies [6][7][8][9][10][11][12][13][14][15]. These biological activities are attributed to their human-health beneficial chemical components, such as polyphenols, carotenoids and other phytochemicals [16,17]. ...
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There is a renewed interest on the reliance of food-based bioactive compounds as sources of nutritive factors and health-beneficial chemical compounds. Among these food components, several proteins from foods have been shown to promote health and wellness as seen in proteins such as α/γ-conglutins from the seeds of Lupinus species (Lupin), a genus of leguminous plant that are widely used in traditional medicine for treating chronic diseases. Lupin-derived peptides (LDPs) are increasingly being explored and they have been shown to possess multifunctional health improving properties. This paper discusses the intestinal transport, bioavailability and biological activities of LDPs, focusing on molecular mechanisms of action as reported in in vitro, cell culture, animal and human studies. The potentials of several LDPs to demonstrate multitarget mechanism of regulation of glucose and lipid metabolism, chemo- and osteoprotective properties, and antioxidant and anti-inflammatory activities position LDPs as good candidates for nutraceutical development for the prevention and management of medical conditions whose etiology are multifactorial.
... Among the Lupinus spp., L. albus (white lupin), L. angustifolius (narrow-leaf lupin), and L. luteus (annual-yellow lupin) are the most consumed [4,5]. Several health-promoting properties have been reported of Lupinus species, mainly L. albus and L. angustifolius, such as antioxidant, anti-inflammatory, hypolipidemic, hypoglycemic, and hypotensive properties among others in several preclinical and clinical human and animal studies [6][7][8][9][10][11][12][13][14][15]. These biological activities are attributed to their human-health beneficial chemical components, such as polyphenols, carotenoids and other phytochemicals [16,17]. ...
... Soybean and lupins are useful to reduce blood cholesterol and thus can protect from hypercholesterolemia and atherosclerosis (Harland and Haffner 2008;Marchesi et al. 2008;Sirtori et al. 2012). Lupins are also considered to have potential for antidiabetic effect (Bertoglio et al. 2011). Phytoestrogens which is obtained from legumes is considered to have positive effects on reducing the risk of cancer and harmful effect on the uterus, thyroid gland and mammary gland (Gierus et al. 2012). ...
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Grain legumes offer many agronomic, environmental and socioeconomic benefits when grown in succession with cereals. They can increase the yields of following crops in the rotation. They fix indirectly atmospheric nitrogen, which makes them economical and environmentally friendly. Globally grain legumes are cultivated on an area of 201,728 thousand ha with a total production of 383,728 thousand tones. In Europe, grain legumes are cultivated on an area of 5726 thousand ha, which represents only 1.8% of total arable lands in Europe. Cultivated area of grain legumes is very low as compared to other words countries and, consequently , Europe imports yearly 20 million tons of soybean meals and 12 million tons of soybean grain. Farmers show lack of interest in cultivating grain legumes due to many climatic, soils, technical, agronomic and economic constraints. These constraints can be removed by technological innovations, provision of more premiums , increasing the sale price and grain yield, and reduction in yield variability of grain legumes.
The link between gut microbiota and obesity or other metabolic syndromes is growing increasingly clear. Natural products are appreciated for their beneficial health effects in humans. Increasing investigations demonstrated that the anti-obesity bioactivities of many natural products are gut microbiota dependent. In this review, we summarized the current knowledge on anti-obesity natural products acting through gut microbiota according to their chemical structures and signaling metabolites. Manipulation of the gut microbiota by natural products may serve as a potential therapeutic strategy to prevent obesity.
The glucose modulating properties of lupin have been attributed to its seed protein γ-conglutin. Here we explored the antidiabetic potential of γ-conglutin purified from lupin seeds in-vitro. To mimic the effects of an orally administered supplement, purified γ-conglutin was hydrolysed by gastrointestinal proteolytic enzymes and the resulting peptides evaluated for their antidiabetic effects in pancreatic β-cells and primary human skeletal muscle myotubes. γ-conglutin peptides did not promote insulin secretion in β-cells but elicited a potent insulin-mimetic action by activating insulin signalling pathways responsible for glycogen, protein synthesis, and glucose transport into myotubes. Additionally, the peptides potently suppressed the activity of DPP4 indicating their potential to increase the half-life of incretin hormones in circulation. These results substantiate the health benefits of consuming lupin seeds as part of a healthy diet and can drive the current market for lupins from primarily stockfeed, towards value-added lupin-based food products for human consumption.
Lupin γ-conglutin beneficially modulates glycemia, but whether it protects against oxidative and lipotoxic damage remains unknown. Here, we studied the effects of γ-conglutin on cell death provoked by hydrogen peroxide and palmitate in HepG2 hepatocytes and insulin-producing MIN6 cells, and if a modulation of mitochondrial potential and reactive oxygen species (ROS) levels was involved. We also investigated how γ-conglutin influences insulin secretion and electrical activity of β-cells. The increased apoptosis of HepG2 cells exposed to hydrogen peroxide was prevented by γ-conglutin, and the viability and ROS content in γ-conglutin-treated cells was similar to that of non-exposed cells. Additionally, γ-conglutin partially protected MIN6 cells against hydrogen peroxide-induced death. This was associated with a marked reduction in ROS. No significant changes were found in the mitochondrial potential of γ-conglutin-treated cells. Besides, we observed a partial protection against lipotoxicity only in hepatocytes. Unexpectedly, we found a transient inhibition of insulin secretion, plasma membrane hyperpolarization, and higher KATP channel currents in β-cells treated with γ-conglutin. Our data show that γ-conglutin protects against cell death induced by oxidative stress or lipotoxicity by decreasing ROS and might also indicate that γ-conglutin promotes a β-cell rest, which could be useful for preventing β-cell exhaustion in chronic hyperglycemia.
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The sample size is the number of patients or other experimental units that need to be included in a study to answer the research question. Pre-study calculation of the sample size is important; if a sample size is too small, one will not be able to detect an effect, while a sample that is too large may be a waste of time and money. Methods to calculate the sample size are explained in statistical textbooks, but because there are many different formulas available, it can be difficult for investigators to decide which method to use. Moreover, these calculations are prone to errors, because small changes in the selected parameters can lead to large differences in the sample size. This paper explains the basic principles of sample size calculations and demonstrates how to perform such a calculation for a simple study design.
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Lupin seed is referred to as an antidiabetic product in traditional medicine. Conglutin-γ, a lupin seed glycoprotein, was found to cause a significant plasma glucose reduction when orally administered to rats in glucose overload trials. Conglutin-γ was identified as being responsible for the claimed biological activity, and the aim of this work was to envisage its hypothetical insulin-mimetic cellular mechanism of action. Insulin is responsible for proteosynthesis control through IRS/AKT/P70S6k/PHAS1 pathways modulation, glucose homeostasis through PKC/Flotillin-2/caveolin-3/Cbl activation and muscle differentiation/hypertrophy via muscle-specific MHC gene transcription control. To assess whether conglutin-γ modulates the same insulin-activated kinases, myoblastic C2C12 cells were incubated after 72 h of differentiation with 100 nM insulin or 0.5 mg/mL (∼10 μM) conglutin-γ. Metformin-stimulated cells were used as a positive control. The effect on the above mentioned pathways was evaluated after 5, 10, 20 and 30 min. In the control cells medium insulin, conglutin-γ and metformin were not added. We demonstrated that insulin or conglutin-γ cell stimulation resulted in the persistent activation of protein synthetic pathway kinases and increased glucose transport, glut4 translocation and muscle-specific gene transcription regulation. Our results indicate that conglutin-γ may regulate muscle energy metabolism, protein synthesis and MHC gene transcription through the modulation of the same insulin signalling pathway, suggesting the potential therapeutic use of this natural legume protein in the treatment of diabetes and other insulin-resistant conditions, as well as the potential conglutin-γ influence on muscle cells differentiation and regulation of muscle growth.
Objectives: Clinical trials with correlated response data based on generalized estimating equations (GEE) have become increasingly popular as they require smaller samples than classical methods that ignore the clustered nature of the data. We have recently derived the recommendation to use the independence estimating equations (IEE) as primary analysis in most controlled clinical trials instead of GEE with estimated correlations [1]. Although several approaches for sample size and power calculation have been proposed, we have shown that most of these procedures are very specific and not as general as required for designing clinical trials. Methods: We extended the previously developed SAS macro GEESIZE to overcome this restriction. Specifically, we have added the option of an independence working correlation matrix required for the IEE. Additionally, we have reformulated the hypotheses to allow for coding that includes an intercept term instead of the previously used analysis of variance coding. Results: To demonstrate the validity of GEESIZE we investigate the calculated sample sizes for specific models where closed formulae are available. For illustration, we utilize GEESIZE for planning a new trial on the treatment of hypertension and thereby exemplify its flexibility. Conclusions: We show that our freely available macro is a very general and useful tool for sample size calculation purposes in clinical trials with correlated data.
A lupin seed glycoprotein, termed γ-conglutin, has previously been found to display insulin-mimetic activity in myocyte models and reduce plasma glucose concentration when orally administered to both rats and humans. To envisage the possible metabolic fate of this bioactive protein, we used in vitro cell and ex vivo tissue models to monitor its transit through the intestinal barrier. Caco-2 cell monolayers and rat intestinal everted sacs were treated with purified γ-conglutin and the protein was immuno-assayed by chemi-luminescence-enhanced Western blotting. The in vitro approach showed that the intact protein can transit from the apical to the basolateral side of the cell monolayers. The unmodified lupin protein was also detected inside the intestinal everted sacs. Proper controls of cell monolayer and sac integrity ruled out the possibility of protein passive leakage.
This review deals with the main proteins of white lupin seed (Lupinus albus, L.) and reports on the current knowledge of the structural and functional properties of these proteins with the aim of providing the first comprehensive, accurate and up-to-date survey on this topic. Lupin seed's four main protein families of globulins, termed α-, β-, γ- and δ-conglutins, are reviewed with specific regard to their molecular and biological features. Their nutritional, technological, nutraceutical and allergenic potentials are also considered. The review is intended to provide nutritionists, food technologists and various stakeholders with the molecular background for a better exploitation of this valuable and accessible protein-rich source.